TIR1/AFB gene family in Brassica juncea var. tumida. In this study, 18 TIR1/AFB genes were identified and could be clustered into six groups. The genes are located in 11 of 18 chromosomes in the genome of B. juncea var. tumida, and similar gene structures are found for each of those genes. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses are found in the promoter of BjuTIR1/AFB genes. The results of qPCR analysis show that most genes have differential patterns of expression among six tissues, with the expression levels of some of the genes repressed by salt stress treatment. Some of the genes are also responsive to pathogen Plasmodiophora brassicae treatment. This study provides valuable information for further studies as to the role of BjuTIR1/AFB genes in the regulation of plant growth, development, and response to abiotic stress.Transport inhibitor response 1/auxin signaling f-box proteins (TIR1/AFBs) play important roles in the process of plant growth and development as auxin receptors. To date, no information has been available about the characteristics of the  TIR1/AFB) family genes perceive the auxin signal as auxin receptors, which then trigger the auxin response signaling pathway [Aux/IAA family genes play important roles in the regulation of auxin response factor genes (ARFs). These ARF family genes are notable for their ability to directly activate or inhibit auxin responsive genes at the transcription level [Auxin is one of most important phytohormones, playing a crucial role in most physiological and biochemical processes of plant growth and development, such as embryo and fruit development, tissue differentiation, root initiation, plant gravitropism, and plant response to biotic and abiotic stresses ,2,3,4. T pathway ,6. It ha pathway ,6; the n pathway . After a pathway . The Auxon level ,10,11. TAt conditions of low auxin concentration in the plant, the Aux/IAA proteins could bind to ARFs, which results in the inhibition of the transcriptional activity of ARFs. When auxin molecules accumulate in plant cells, the reaction between the auxin molecules and TIR1/AFBs triggers a combination of TIR1/AFBs and Aux/IAAs, leading to degradation of the Aux/IAA proteins through the ubiquitin-26S proteasome system. After the degradation of Aux/IAA proteins, the released ARFs could effectively activate a form of auxin response system gene, which attempts to correctly regulate the plant’s response to auxin signaling .Arabidopsis, which includes an F-box domain in the N terminus and several Leucine-rich repeat (LRR) domains in the C terminus [tir1 loss-of-function mutants are deficient in some auxin-regulated growth processes, such as hypocotyl elongation and lateral root formation [TIR1 complex by binding directly to SCFTIR1; TIR1 is identified as an auxin receptor [AFB1, AFB2, and AFB3 were identified, playing similar roles to TIR1 in the same manner [TIR1, AFB1, AFB2m and AFB3 play roles in the plant response to auxin in the root, TIR1, AFB2, and AFB3 contribute equally in this process, while AFB1 does not assemble into an SCF complex as efficiently [afb1, afb2, and afb3 seedlings all performed with a similar phenotype to the wild type, even in double mutant combinations involving the afb mutants and tir1; no obvious development defects have been found. However, severe development defects in cotyledon, hypocotyl elongation, and gravitropism have been observed in triple and quadruple mutants [afb4 loss-of-function mutants [afb4 and afb5 are resistant to the synthetic auxin picloram but not IAA or 2,4-D, indicating that these two proteins are selective for picloram [TIR1 was the first identified in terminus . The tirormation . Overexpormation . The TIRreceptor ,6. Shorte manner . It is wiciently ,15. For  mutants . Both afpicloram ,17,18.Arabidopsis thaliana, overexpression of AtTIR1 has resulted in overproduction of lateral roots [GmTIR1 and GmAFB3 genes resulted in fewer nodule numbers in soybean [AtAFB3 plays important roles in lateral root production, in the manner of specially responding to nitrate treatment [afb4 loss-of-function mutant, the number of lateral roots is significantly less than that in wild-type seedlings, indicating that AFB4 is also involved in lateral root development in Arabidopsis [afb4 mutant had other defects in the development process, such as shorter hypocotyls, lower height, and delayed flowering [Arabidopsis, lateral root production is normally inhibited by ABA treatment—however, overexpression of AtAFB2 has been shown to counteract this defect, indicating a response pathway from abiotic stress to this particular gene [Arabidopsis, the transcriptional levels of AtTIR1, AtAFB1, AtAFB2, and AtAFB3 are reduced by a flagellin-derived, 22-amino acid peptide , resulting in the repression of auxin signaling in the plant. Repressed auxin sensitivity could restrict growth of pathogens, further highlighting the crucial roles of TIR1/AFBs in plant tolerance to abiotic stresses [IAA has been found to induce main root growth inhibition rapidly, while the TIR1/AFB’s auxin receptor mutants failed to display this effect ,20. In Aal roots . Similar soybean . It has reatment . In the bidopsis . Besideslowering . The TIRlar gene . In Arabstresses .Brassica juncea var. tumida Tsen et Lee) is an important crop in China, with great economic benefits. How to improve the yield of this crop is a key issue for the Chinese pickle industry. Auxin plays pleotropic roles in plant growth and development, exploring whether or not the effect of the auxin signaling pathway in crop production is meaningful. TIR1/AFB receptors also exist in tumorous stem mustard; however, no related report has been found for the function of those genes in this crop. Here, we have identified the TIR1/AFB family members in tumorous stem mustard, and analyzed their genes and the protein characteristics of those genes, as well as their expression patterns during plant growth and development. This study will be useful as a starting point for more functional investigations of TIR1/AFBs in tumorous stem mustard.Tumorous stem mustard (Plasmodiophora brassicae (OD600 = 0.07) for the indicated time points.In this study, tumorous stem mustard cultivar Yong An XiaoYe was used for gene expression pattern analysis. The seeds were sowed into 2:1 vermiculite:turfy soil, cultured under constant light at 22 °C with a 16/8 h light/dark regime in culture room. For the salt stress assay, the seeds of Yong An Xiao Ye were sowed in pots in a culture room, and two-week-old seedlings were irrigated by nutrient solution with or without 200 mM NaCl for 3, 6, 12, 24 h, respectively, and the roots were collected for real-time quantitative PCR analysis. For pathogen treatment, two-week-old seedlings were then inoculated with a 5 mL resting spore suspension of TIR1, AFB1, AFB2, AFB3, AFB4, and AFB5 sequence of A. thaliana were obtained from The Arabidopsis Information Resource website (https://www.arabidopsis.org/). Using the TIR1/AFB genes as the query sequence to blast relative homologs in the B. juncea genome database (http://brassicadb.org/brad/). The gene structure diagram was drawn using the online software of the GSDS2.0 server (http://gsds.cbi.pku.edu.cn/), the phylogenetic analysis was done using MEGA5 software [The software  with thehttps://web.expasy.org/compute_pi/), and the protein secondary structure was analyzed by SMART (http://smart.embl-heidelberg.de/) and CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The diagram of the protein domain was drawn by ExPASy. The cis-elements in the promotor of TIR1/AFBs were predicted by SOGO online software (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace).The amino acid isoelectric point and molecular weight were predicted by online ExPASy software . The total RNA samples were treated with DNase I  to remove contaminating genomic DNA. First-strand cDNA was synthesized from the total RNA using a FastQuant RT Kit (Tiangen Biotech (Beijing) Co., Ltd.). Real-time qPCR (qRT-PCR) was performed using SuperReal PreMix Plus  Co., Ltd.).  qRT-PCR , and theAll data were analyzed using SigmaPlot 10.0  and SPSS 16.0 software. The averages and standard deviations of all results were calculated, one-way ANOVA followed by the Dunnett test was used.TIR1/AFB genes in B. juncea var. tumida; they were named BjuTIR1 and BjuAFBs, and the CDS sequences of those genes can be found in BjuO006283 contig, which could not be anchored in the chromosome of B. juncea var. tumida, the left 17 BjuTIR1/AFBs genes located are in 11 of 20 B. juncea chromosomes. Each of the chromosome A03, A09, and B06 contains three genes, and the other eight genes were located at different chromosomes  [cis-elements related to plant response to biotic-stresses, such as GT1GMSCAM4 (GAAAAA) [cis-elements MYCCONSENSUSAT (CANNTG) [BjuTIR1/AFBs genes. Those cis-elements were involved in plant response to dehydration stress. Some cis-elements responsive to low temperatures were also found, such as LTRE1HVBLT49 (CCGAAA) [cis-elements responsive to ABA   and GMSA(TGTCTC)    and GCCC(GAAAAA) . The cis(CANNTG) , MYB1AT (CANNTG) , and MYB(CANNTG)  were als(CCGAAA) , CRTDREH(CCGAAA) , and LTR(CCGAAA) . BesidesELATERD1  and ABREELATERD1 ) and salELATERD1  were alsREATRE22 . This reBjTIR1/AFB family genes; the raw data of the qPCR results can be found in the BjuTIR1A, BjuTIR1D, and BjuAFB1B were highly expressed in most tissues, and moderate expression levels were found for BjuTIR1C, BjuAFB1B, BjuAFB3A, and BjuAFB3C  for 6, 12, 24, 48, and 72 h, respectively, and the roots were collected for (RT-qPCR) analysis. The results showed that BjuTIR1A and BjuAFB3B were severely induced by P. brassicae treatment; BjuTIR1D, BjuTIR1E, BjuAFB1A, BjuAFB1B, BjuAFB2, BjuAFB3D, BjuAFB4, and BjuAFB5B were induced moderately by P. brassicae treatment . The comparable auxin receptor gene numbers in the A or B sub-genome indicates that the genome of B. juncea var. tumida experienced co-linearity in the gene duplication process. Most of the auxin receptor genes contained three exons and two introns, the exceptions being BjuAFB1D and BjuAFB1D, both of which contained four exons and three introns (lication . In A. tentified ,16,17,18a genome . The mul introns . The genOsTIR1 and OsAFB2 in rice [BjuAFB2 was low in all tissues of B. juncea var. tumida (BjuAFB2 might not play an important role as a main auxin receptor. In contrast, the high expression levels of BjuTIR1A and BjuTIR1D indicate that these two genes might be the main auxin receptors in B. juncea var. tumida. According to the results of the gene expression pattern, tissue-specific gene expressions were identified, such as BjuTIR1A demonstrating influence on the regulation of flower development, and BjuTIR1F may specifically influence pod development regulation.Abnormal phenotypes of enlarged flag leaf inclination, more tillers, early flowering and a lower tolerance to salt were found due to reduction of  in rice ,45. Howe. tumida , suggestAtmiR393a could repress the expression levels of auxin receptors like TIR1, leading to the inhibition of plant growth and enhanced resistance to salt stress [BjuTIR1/AFB family genes under salt treatment were consistent with the previous study. The exception was BjuTIR1A—the expression of this gene was significantly induced by salt treatment, which might result from feedback of the signaling transduction.It has been reported that auxin signaling plays an important role in plant resistance to abiotic stress, especially at high salinity . In thist stress . The redZea mays defense against Rhizoctonia solani [Arabidopsis resistance to the bacterium Pseudomonas syringae [Arabidopsis, AtTIR1 and AtAFB1 are transcriptionally upregulated in clubroots, and the mutants of tir1, afb1-3, and afb1-3 afb2-3 all resulted in more susceptible reactions to the root pathogen P. brassicae [BjuTIR1A was severely induced by P. brassicae treatment, especially at 6 and 12 h after inoculation. Besides that, all the BjuTIR1s were induced by this pathogen treatment, except for BjuTIR1B (BjuAFB1 genes, the expression levels of AFB1A, AFB1B, and AFB1C also increased at 48 h after pathogen inoculation (BjuTIR1s and BjuAFB1s might play similar roles to AtTIR1/AFB1s in the processes of plant defense against P. brassicae invasion.The auxin signaling pathway mediated by TIR1/AFBs is usually involved in plant resistance or tolerance to pathogen attacks, such the anthracnose disease in cassava , Zea maya solani , or Arabsyringae . In Arabrassicae . In thisBjuTIR1B . For Bjuculation . The indBjuTIR1/AFB genes were identified, and they have similar gene structures and protein domains to the auxin receptors of Arabidopsis. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses were found in the promoter of BjuTIR1/AFB genes, indicating those genes may play a role in the regulation of plant development and defense against biotic and abiotic stresses, which could benefit from a more targeted follow-up study. Gene expression analysis showed that some BjuTIR1/AFB family genes exhibit a special expression pattern, and most genes are responsive to salt stress treatment. Together, our data provides a useful foundation for future research regarding the function of auxin receptors in B. juncea var. tumida.In this study, a total of 18


This study aimed to investigate the correlation of ultrasonography (US) of synovitis with disease activity and clinical response to etanercept (ETN) in juvenile idiopathic arthritis (JIA) patients. Eighty-two JIA patients who underwent ETN treatment for 24 weeks were consecutively enrolled. US evaluations of 28 joints  at baseline were performed using grey-scale US and power doppler (PD) US, and US synovitis was defined as grey-scale abnormalities or PD abnormalities. Clinical response was assessed according to the ACRpedi 50 response criteria. In total, 2296 joints were scanned and 608 (26.5%) joints presented US synovitis, which was numerically higher than clinical synovitis (513 (22.3%)). The mean number of joints showing synovitis on US was 7.42±3.35, which was also numerically higher than that of clinical synovitis (6.26±2.70). The number of joints showing synovitis on US was positively correlated with C-reactive protein, erythrocyte sedimentation rate, number of joints with active disease, number of joints with limited range of motion, physician's global assessment of disease activity, parent/patient global assessment of overall well-being, and childhood health assessment questionnaire score. Most interestingly, the baseline number of joints showing synovitis on US was increased in ACRpedi 50 response JIA patients compared to non-response JIA patients, and it serves as an independent predictive factor for higher clinical response to ETN treatment. In conclusion, US is a more sensitive test to evaluate subclinical synovitis and disease activity in JIA patients, and US synovitis might serve as a marker for predicting increased clinical response rate to ETN treatment. Juvenile idiopathic arthritis (JIA), as a chronically inflammatory and autoimmune disease, is a critical health issue affecting children and adolescents worldwide ,2. The iUltrasonography (US), initially applied and recommended for evaluation of subclinical synovitis, disease activity, and treatment response in rheumatoid arthritis, has been introduced for JIA examination recently ,8. US prEighty-two JIA patients from the Children's Hospital of Nanjing Medical University were consecutively enrolled in this study from January 2015 to November 2017. The inclusion criteria were: 1) diagnosed as JIA according to the International League of Associations for Rheumatology criteria ; 2) abouAfter enrollment, patients' characteristics were recorded including: 1) demographic features: age, gender, height, weight, and disease duration; 2) JIA subtypes: oligoarthritis, rheumatoid factor (RF) negative-polyarthritis, RF-positive polyarthritis, systemic, enthesitis-related arthritis, or psoriatic; 3) clinical features: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), joints with active arthritis, joints with limited range of motion, physician's global assessment of disease activity, parent/patient global assessment of overall well-being, and childhood health assessment questionnaire (CHAQ) scores; 4) initiation of treatment: ETN, methotrexate (MTX), leflunomide (LEF), and other disease-modifying antirheumatic drugs (DMARDs).US evaluation of 28 joints , proximal interphalangeal of hands (PIP), and knee) at baseline were performed for evaluation of the disease and its potential to predict response to ETN in all patients (n=82). US was done with MyLab 70  with a 6–18 MHz linear probe and a Logiq S8  with an 8–18 MHz linear probe according to the settings described in a previous study , which wAll 82 JIA patients received ETN treatment for 24 weeks as subcutaneous injections, twice weekly, at the dose of 0.4 mg/kg. According to the disease conditions, MTX, LEF, or other DMARDs were combined.After the 24-week treatment, clinical response was assessed according to the ACRpedi 50 response criteria: 50% improvement from baseline in at least 3 of any 6 variables of the JIA core sets, with no more than 1 variable worsening by more than 30% . The 6 vt-test, correlation between two variables was assessed by Pearson’s test, and factors affecting clinical response were assessed by univariate and multivariate logistic regression analysis. A P<0.05 was considered significant.Statistical analysis was performed using SPSS 22.0 software . Data are reported as means±SD or count (percentage). Comparison between two groups was done by The mean age of the enrolled 82 JIA patients was 6.8±2.8 years with 27 males and 55 females. The disease duration was 2.78±1.74 years. There were 25 (31%) oligoarthritis, 25 (31%) RF-negative polyarthritis, 12 (15%) RF-positive polyarthritis, 10 (12%) systemic arthritis, 8 (10%) enthesitis-related arthritis, and 2 (2%) psoriatic JIA subtypes. The mean CRP and ESR were 38.5±23.0 mg/L and 32.7±20.0 mm/h, respectively. The other detailed characteristics of JIA patients are listed in The detection rate of US synovitis was numerically higher compared to the detection rate of clinical synovitis regarding shoulder, elbow, wrist, MCP, PIP, and knee joints at baseline; in a total of 2296 scanned joints, 608 (26.5%) joints presented US synovitis, which was numerically higher compared to 513 (22.3%) joints with clinical synovitis . Also, tThe number of joints showing synovitis on US at baseline was positively correlated with CRP P<0.001, , ESR P=, number After the 24-week ETN treatment, 58 (70.7%) patients achieved ACRpedi 50 response while 24 (29.3%) patients failed . The numUnivariate logistic regression model revealed that the number of joints showing synovitis on US at baseline  and CRP  predicted higher ACRpedi 50 response, while systemic JIA subtype  predicted lower ACRpedi 50 response . Only faIn the 39 patients who underwent US detection after 24-week ETN treatment, the number of joints showing synovitis on US was greatly reduced (4.01±1.68) compared to baseline (7.29±3.14) (P<0.001). Also, mean number of joints showing clinical synovitis was also decreased at 24 weeks (2.85±1.53) compared to baseline (6.14±2.15) (P<0.001).In the current study, we found that in JIA patients who underwent ENT treatment: 1) US synovitis occurrence was 26.5%, and the detection rate was higher compared to clinical synovitis; 2) US synovitis was correlated with increased disease activity; 3) US synovitis predicted higher clinical response to ETN treatment.The detection and management of synovitis, as a chief factor responsible for inflammation and structural joint damage in JIA, has attracted great attention . TraditiUS synovitis also has potential to be used as a disease-monitoring marker in arthritis disease. For instance, a study detected US synovitis of wrist joints in 50 early rheumatoid arthritis patients, and disclosed PD-US synovitis showed positive correlation with TNF-α, IL-6, and angiopoietin-1 and -2 levels . AnotherApart from the above-mentioned application of US in synovitis, it may serve as a potential marker for disease prognosis as well in several types of arthritis. A study with 141 rheumatoid arthritis (RA) patients who underwent synthetic and biologic DMARDs treatments for 12 months underwent wrist, MCP 2/3, PIP 2/3, and metatarsophalangeal 2/5 gray scale US and PD-US found that lower DAS28 score and PD-US score correlated with higher clinical remission after treatment . AnotherIn the present study, we found that the baseline number of joints showing synovitis on US was correlated with increased response in JIA patients, serving as an independent predictive factor for higher clinical response rate to ETN treatment. The possible explanations are: 1) increased number of joints showing synovitis on US correlates with higher disease inflammation and activity, and ETN is a target anti-inflammation agent, thus ETN presented better efficacy in these high-inflammation patients; 2) clinical treatment in our study was evaluated using ACRpedi 50 response criteria , which uSeveral limitations are present in this study: 1) due to patients' unwillingness, US examinations after treatments were only done in a few patients, thus the effect of ETN on US synovitis attenuation was not analyzed in our study; 2) the sample size of this study was relatively small because of the high cost of ETN; 3) most patients in this study came from Middle China, thus multiple-region and multiple-center validation is needed in the future; 4) a commonly affected joint, the ankle, was not included in the US evaluation in this study, which should be further explored in the future.In conclusion, US is a more sensitive test to evaluate subclinical synovitis and disease activity in JIA patients, and US synovitis might serve as a marker for predicting increased clinical response rate to ETN treatment.


Euglycemic diabetic ketoacidosis (euDKA) related to sodium-glucose cotransporter 2 inhibitor (SGLT2-I), despite being reported as consistent, though infrequent, adverse effect in all trials on SGLT2-I in type 2 diabetes mellitus (T2D), still remains poorly known in the real world. On the other hand, the use of this new class of antihyperglycemic agents is expected to increase based on the recent solid evidence of remarkable cardiorenal protection. Therefore, improving awareness on risk factors, diagnosis, and treatment of euDKA is essential to allow correct implementation of SGLT2-I in clinical practice. We here report a T2D patient admitted to the emergency department and then transferred to the nephrology-dialysis unit because of severe euglycemic diabetic ketoacidosis (euDKA) related to sodium-glucose cotransporter 2 inhibitor (SGLT2-I). In our patient, a concurrent acute kidney injury at presentation, initially attributed to excessive use of nonsteroid anti-inflammatory agents, and the absence of severe hyperglycemia led to delayed diagnosis and proper therapy. The detailed description of decision-making process for diagnosis and therapy, and the analysis of precipitating factors as well, discloses the helpful contribution of nephrologist to optimize prevention and management of euDKA. Diabetic ketoacidosis (DKA) is a major life-threatening event of type 1 diabetes mellitus that can, however, occur in type 2 (T2D) in the presence of relative insulin deficiency. A subtype of DKA, labeled “euglycemic” (euDKA), is characterized by plasma bicarbonate ≤10 mEq/L and glycemia ≤300 mg/dL . The int2. In the last month, due to worsening glycemic control, the diabetologist prescribed a lower intake of calories and added empagliflozin 25 mg/day on top of usual treatment (metformin 1700 mg/day). The new therapy led to 14-kg body weight loss (from 67 to 53 kg). Family also mentioned bis-in-die therapy with nonsteroid anti-inflammatory agents (NSAID) for a joint pain throughout the two weeks before hospitalization. At presentation, BMI was 21.5 kg/m2. Vital signs were temperature 37.2 °C, pulse rate 110 beats/min, blood pressure (BP) 90/60 mmHg, respiratory rate 40 breaths/min, and urinary output 100 mL/h. Physical examination showed dehydration and no relevant findings at objective examination of lungs and abdomen. A 67-year-old woman with a 5-year history of T2D presented to the emergency department (ED) of a public hospital because of abdominal pain and impaired conscious level , occurring after one week of fever, malaise, and dyspnea. Family reported that the patient was not a smoker nor was used to drink alcohol and had normal blood pressure. Clinical charts showed that she was free of cardiovascular disease and had albuminuria (800–1100 mg/g creatinine) with estimated glomerular filtration rate (eGFR) 75–85 mL/min/1.73 mIn ED, diagnosis was “metabolic acidosis and hyperkalemia in diabetic patient with acute kidney injury (AKI) likely related to NSAIDs”, and treatment was intravenous (iv) bolus (140 mmol) of sodium bicarbonate 1.4% followed by continuous iv infusion of normal saline 0.9% and sodium bicarbonate 1.4% at the rate for either solution of 125 mL/h. In the subsequent 6 h, acidosis status slightly improved, thus leading to reduction of bicarbonate rate to 20 mL/h.After 12 h of infusion, a renal consultant was called because of hypernatremia onset and persistent acidosis. Nephrologist asked for urinalysis, not included in the initial screening, that disclosed very high glucose and ketones levels. The consultant diagnosis was “AKI and DKA related to SGLT2-I”. Therefore, SGLT2-I therapy and bicarbonate infusion were discontinued, iv infusion of Lispro, a rapid-acting insulin analog (0.1 UI/kg/h) was started, and KCl 20 mmol/h was added to the normal saline infusion. In the same day, due to severity of acidosis and hypernatremia onset coupled with worsening of hemodynamics (BP 80/60 mmHg) and mental status, the patient was transferred to the nephrology department. She underwent 4-h treatment with bicarbonate hemodialysis for two consecutive days with ultrafiltration rate adjusted to allow volume expansion of 2.0 L/session. After the first session, urinary output increased up to about 500 mL/h due to repletion of extracellular volume, concomitant increase of BP (130/60 mmHg), and persistent glycosuria. Within few days, a significant improvement was observed in mental status, systemic hemodynamics, renal function, and hydroelectrolyte homeostasis. Insulin infusion continued for one week. Additional lab exams obtained in the nephrology ward demonstrated mycotic and bacterial infection of urinary tract associated with high procalcitonin  that was efficaciously treated by culture-guided therapy. Lab tests also showed high glycated hemoglobin (HbA1c 12%), very low fasting C-peptide (0.43 ng/mL), and absence of autoantibodies against insulinoma-associated protein 2 (IA-2 6.3 U/mL) and glutamic acid decarboxylase (GAD 4 U/mL) suggestive for latent autoimmune diabetes in adults (LADA). Following the complete resolution of the clinical picture, a kidney biopsy was performed at day 15 because of high albuminuria (942 mg/g) not readily attributable to diabetic nephropathy according to the relatively short duration of T2D and the absence of diabetic retinopathy. Histopathology, however, revealed overt diabetic nephropathy .The patient was discharged after recovery of renal function, volemia, and metabolic control. Diagnosis was “euDKA related to SGLT2-I associated with AKI due to osmotic diuresis-induced volume depletion in patient with type 2 diabetic nephropathy”. The patient was switched on chronic insulin therapy and regular nephrology follow-up was indicated.In our patient, the whole picture was coherent with euDKA due to SGLT2-I .2 and rarely associates with serum bicarbonate <14 mmol/L [However, diagnosis was initially missed mainly due to the absence of urinalysis at admission. Indeed, the moderate hyperglycemia in euDKA has the deleterious effect of obscuring diagnosis as compared with “traditional DKA” characterized by severe hyperglycemia ,10,11,124 mmol/L . On the 4 mmol/L . Besides4 mmol/L . (4) TheIn conclusion, better knowledge of risk factors, diagnosis, and treatment of euDKA likely represent an essential step to translate the cardiorenal protective efficacy of SGLT2-I proven in RCTs  into ful


Phyllomedusa sauvagii skin secretion-derived cDNA library, and thereafter, the presence of the mature peptide, namely dermaseptin-PS3 (DPS3), was confirmed by LC–MS/MS. Moreover, this naturally occurring peptide was utilized to design two analogues, K5, 17-DPS3 (introducing two lysine residues at positions 5 and 17 to replace acidic amino acids) and L10, 11-DPS3 , improving its cationicity on the polar/unipolar face and hydrophobicity in a highly conserved sequence motif, respectively. The results in regard to the two analogues show that either increasing cationicity, or hydrophobicity, enhance the antimicrobial activity. Also, the latter analogue had an enhanced anticancer activity, with pretreatment of H157 cells with 1 µM L10, 11-DPS3 decreasing viability by approximately 78%, even though this concentration of peptide exhibited no haemolytic effect. However, it must be noted that in comparison to the initial peptide, both analogues demonstrate higher membrane-rupturing capacity towards mammalian red blood cells.The skin secretions of the subfamily Phyllomedusinae have long been known to contain a number of compounds with antimicrobial potential. Herein, a biosynthetic dermaseptin-precursor cDNA was obtained from a  Of the many anuran skin-derived peptides that are known, dermaseptin and dermaseptin-like peptides are the most remarkable candidates for developing new antibiotics in Hylidae frogs ,2,3. AltNumerous studies indicate that the net charge is a key factor influencing the binding of AMPs to membranes, as AMPs bind to the membrane by electrostatic interaction and competitively replace the divalent cation ,10,11. TPhyllomedusa sauvagii using a combination of shotgun cloning and mass spectrometry. The corresponding chemically synthesised replicate exerted weak antibacterial activity towards pathogenic microorganisms and weak cytotoxic activity towards tumour cells. Therefore, we designed two analogues of this naturally occurring peptide, K5, 17-DPS3 and L10, 11-DPS3, to potentially optimize its cationicity on the polar/unipolar face and hydrophobicity in conserved sequence motif of dermaseptin, respectively. Herein, we describe the discovery of a biosynthetic precursor, preprodermaseptin, encoding an antimicrobial peptide, DPS3, from the skin secretion of Phyllomedusa sauvagii. More specifically, the domain architecture of this preprodermaseptin transcript   . . In particular, K5, 17-DPS3 displayed MIC values of 8 µM or less against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, as well as yeast . The parent peptide, DPS3, generally showed weak antimicrobial activity, although it did exhibit better activity against Gram-negative bacteria. As expected, when compared to the parent peptide, both artificial analogues displayed enhanced antimicrobial activity against all the microorganisms examined . In part50 value more than 10-fold lower than either of the other peptides  and positively charged AMPs is an essential step allowing the cationic peptides to selectively aggregate on the bacterial membrane; therefore, it is also believed that changing the number of positive charges present in an AMP can likely change its membrane binding ability [10, 11-DPS3, but less haemolysis. Similarly, K4K20-S4, a dual lysine-substituted dermaseptin-S4, also shown increased antimicrobial activity, along with two-fold haemolytic potency and higher lipophilic affinity [Early research has revealed a strong correlation between α-helical domain and antimicrobial activity, involving the local fusion of the membrane leaflets, pore formation, cracks, as well as the depth of membrane insertion ,25,26. A ability ,10,11. A ability ,28. We saffinity . Previou10, 11-DPS3 exhibited an enhanced antiproliferative impact. Recent studies have shown that cancer cell membranes, similar to the bacterial membrane, carry a negative charge due to overexpression of anionic molecules, such as phosphatidylserine, sialic acid and glycosaminoglycans (GAGs) [10, 11-DPS3 (1 µM) decreased by approximately 78%, whilst no membrane lysis was observed in mammalian erythrocytes exposed to the same concentration of peptide, possibly suggesting that a nonlytic mechanism may be involved, at least at lower concentrations. At higher peptide concentrations (>1 µM), the higher anticancer cell impact is consistent with increasing haemolysis activity, suggesting that this is possibly due to cell membrane disruption. However, further investigations will be needed to confirm this further.DPS3, and its analogues, all exhibit antiproliferative effects on the tested cancer cells, although Ls (GAGs) ,17,18. Ts (GAGs) ,19,22. A5, 17-DPS3 and L10, 11-DPS3 both exhibited more potent antimicrobial activity, whilst L10, 11-DPS3 also exhibited enhanced anticancer activity, even at concentrations that had minimal impact upon healthy mammalian cells. This would suggest that K5, 17-DPS3 and L10, 11-DPS3 may have promise as antimicrobial agents, and L10, 11-DPS3 could have potential as an anticancer agent. However, further investigations will be required to determine the exact mode of action that these peptides utilise against the microbial and cancer cells.The 23-mer peptide DPS3 reported here is relatively short in comparison to other naturally occurring dermaseptins, which are commonly 28 to 34 amino acids. Although DPS3 exhibited relatively weak bioactivity for dermaseptin family members, we rationally designed two DPS3 analogues with the aim of improving its bioactivity. In particular, KPhyllomedusa sauvagii (4–6 cm snout-to-vent length) obtained from a commercial source in the United States were exposed to 12 h of light at 20–25 °C daily, and multivitamin-loaded crickets were provided as the fodder three-times/week. Following four-month breeding, dermal secretions were collected via surface electrical stimulation [Three specimens of mulation . In summPhyllomedusa camba was reverse-transcript to the cDNA library. The 3′-RACE was conducted using the cDNA library and the primer (5′-ACTTTCYGAWTTRYAAGMCCAAABATG-3′) that was designed to a segment of the 5′-untranslated region of cDNAs from Phyllomedusa species . The RACE products were subjected to purification and cloned, and finally sequenced by the ABI 3100 Automatic Capillary Sequencer.The shotgun cloning employed the rapid amplification of cDNA ends (RACE) technique with a degenerate primer, which has been described previously . BrieflyThe identification of DPS3 from the skin secretion was performed according to our previous study . Briefly5, 17-DPS3 (ALWKKILKNAGKAALNKINQIVQ-NH2) and L10, 11-DPS3 (ALWKDILKNLLKAALNEINQIVQ-NH2). The mean hydrophobicity, hydrophobic moment and helical wheel projections of peptides were predicted by Heliquest (http://heliquest.ipmc.cnrs.fr/). Peptide synthesis was carried out using Tribute peptide synthesizer  along with Rink amide resin and standard Fmoc chemistry, which was described in a previous study [To investigate the effect of the increasing cationicity and hydrophobicity, the peptide DPS3 was used as the framework to design two analogues where the two acidic amino acids (at positions 5 and 17) and two neutral amino acid residues (at positions 10 and 11) were substituted with lysine and leucine residues, respectively. Accordingly, theses analogues are named Kus study . http://bestsel.elte.hu [The secondary structure was determined by a CD spectrometer . The analysis method was performed as previous study . The obt.elte.hu ) to calcS. aureus (NCTC 10788), E. coli (NCTC 10418) and C. albicans (NCYC 1467) [The antimicrobial activity of three peptides was evaluated via the minimal inhibitory concentrations (MICs) against YC 1467) . Microor−4 to 10−9 M. After adding MTT, the formazan crystals were dissolved and read by the plate reader at 570 nm.Cell viabilities were achieved using a typical MTT assay . BrieflyThe haemolytic activity of each peptide was determined using a 2% suspension of horse blood cells  as previous study . The red


In 20% of meningeal tuberculosis cases, the cerebrospinal fluid cell count is between 500 and 1,500/mm3, and in 65% between 100 and 500/mm3.1A 16-year-old boy was admitted to a rural hospital in the Democratic Republic of Congo with a 1-month history of abdominal pain, headache, and nightly fevers, and a 1-week history of seizures. Physical examination included general weakness, low-grade fever (37.5°C), neck rigidity, and bilateral abducens palsy leading to double vision , typical2 and explaining the abdominal pain. Simultaneous pulmonary tuberculosis or human immunodeficiency virus (HIV) infection were ruled out by chest X-ray and a HIV rapid test.In addition, physical examination revealed spinal tenderness at T 10-11 with normal reflexes, sensation, and strength of lower extremities bilaterally. Abdominal and cardiopulmonary examination was unremarkable. Spinal X-ray showed destructive anterior spondylodiscitis at T 10-11 with a paravertebral abscess , again tDue to pulmonary tuberculosis already experienced as an infant and current extrapulmonary manifestation, an extended treatment scheme with rifampicin, isoniazid, ethambutol, pyrazinamide, and streptomycin (2SRHZE/RHZE/9RH), as well as initially beginning with corticosteroids (prednisone 1 mg/kg) was chosen.3. One month later, the patient was back to normal daily life, playing football with friends.Within 2 weeks, the abducens palsy was resolved completely, the abdominal pain improved, and the spinal fluid cell count dropped to 23 cells/mm3Tuberculosis is still endemic in many low- and middle-income countries such as the Democratic Republic of Congo with a tuberculosis incidence of 323 cases per year per 100,000 inhabitants and a high number of undetected cases every year.This case illustrates that a good clinical examination, knowledge on the epidemiological background, and basic laboratory technology may allow diagnosis and successful treatment even without modern technologies such as polymerase chain reaction and magnetic resonance imaging/computed tomography scan in resource-limited settings.


Motivation: Mass cytometry is a technique used to measure the intensity levels of proteins expressed by cells, at a single cell resolution. This technique is essential to characterize the phenotypes and functions of immune cell populations, but is currently limited to the measurement of 40 cell markers that restricts the characterization of complex diseases. However, algorithms and multi-tube cytometry techniques have been designed for combining phenotypic information obtained from different cytometric panels. The characterization of chronic HIV infection represents a good study case for multi-tube mass cytometry as this disease triggers a complex interactions network of more than 70 cell markers.Method: We collected whole blood from non-viremic HIV-infected patients on combined antiretroviral therapies and healthy donors. Leukocytes from each individual were stained using three different mass cytometry panels, which consisted of 35, 32, and 33 cell markers. For each patient and using the CytoBackBone algorithm, we combined phenotypic information from three different antibody panels into a single cytometric profile, reaching a phenotypic resolution of 72 markers. These high-resolution cytometric profiles were analyzed using SPADE and viSNE algorithms to decipher the immune response to HIV.Results: We detected an upregulation of several proteins in HIV-infected patients relative to healthy donors using our profiling of 72 cell markers. Among them, CD11a and CD11b were upregulated in PMNs, monocytes, mDCs, NK cells, and T cells. CD11b was also upregulated on pDCs. Other upregulated proteins included: CD38 on PMNs, monocytes, NK cells, basophils, B cells, and T cells; CD83 on monocytes, mDCs, B cells, and T cells; and TLR2, CD32, and CD64 on PMNs and monocytes. These results were validated using a mass cytometry panel of 25 cells markers.Impacts: We demonstrate here that multi-tube cytometry can be applied to mass cytometry for exploring, at an unprecedented level of details, cell populations impacted by complex diseases. We showed that the monocyte and PMN populations were strongly affected by the HIV infection, as CD11a, CD11b, CD32, CD38, CD64, CD83, CD86, and TLR2 were upregulated in these populations. Overall, these results demonstrate that HIV induced a specific environment that similarly affected multiple immune cells. Cytometry is an experimental technique used to measure the intensity levels of proteins expressed by cells, at a single cell resolution. However, none of these technologies can currently handle the simultaneous study of more than 50 markers . This reBioinformatics approaches have been successfully developed and applied to extend the number of observable cell markers per cytometric profiles through the merging of phenotypic information obtained from different cytometry profiles (generated from different cytometry panels). In such approaches, phenotypic information from the different profiles is combined into a single profile based on the expression of a common set of shared markers –4. EspecHIV infection is still a major sexually transmissible disease. Although detected by different immune receptors, HIV is not cleared by the immune system. A global overview of the cell interaction network triggered in HIV infections is required to understand the immune system dysfunctions and to set up new therapeutic treatments.+ T cells. In the long term, HIV induces a decrease of T cells number both in the blood and in the lymphoid organs. This situation then triggers a severe immune deficiency, that leads to Acquired Immunodeficiency Syndrome (AIDS) , which is to the detriment of CD14high CD16low monocytes  , 7. The onocytes . Non-claonocytes –11. In aonocytes . Furtheronocytes , 14. Intonocytes . This exonocytes . OverallMany proteins involved in chronic inflammation in HIV-infected subjects have been described , 20. IndHere, we used the CytoBackBone algorithm  to charaThis experiment was approved by the Comité de Protection des Personnes (CPP) Ile de France VII, under protocol number PP 14-003. All subjects gave written informed consent to participate in this study.n = 3) and HIV-1 ART-treated non-viremic donors  was collected in lithium heparin tubes by the Etablissement Français du Sang  and Hôpital du Kremlin Bicêtre, respectively. Information concerning the gender, current age, contamination pathway, viral load, year of detection of the HIV infection, starting year of ARV treatment, and the type and the duration of treatment is provided for each HIV-infected patient in Whole blood from healthy (n = 6), and six new healthy subjects (n = 6) was collected. Information concerning the gender and the current age is provided for each HIV-infected patient and each healthy donor in To validate the results with a single cytometric panel, blood from the three previous HIV-infected subjects, three new HIV-infected patients  and contained 18.5% glycerol  in 1X-Dulbecco's phosphate buffered saline (DPBS), without CaCl2 or MgCl2, pH 7.4 . This solution allowed freezing and recovery of all blood leukocytes, especially polymorphonuclear cells, which are highly labile and cryopreservation-sensitive. Healthy and HIV-infected samples used for the multi-tube 72-marker experiment were cryopreserved for a maximum of 12 days.Blood samples were processed according to a previously described protocol . The cel6 cryopreserved fixed cells were washed twice with staining buffer  and labeled with conjugated antibodies according to the following procedure. Cells were incubated at 4°C for 30 min with a mix of the metal-labeled surface antibodies (Abs) in staining buffer. After two washes with 1X DPBS, cells were incubated in fixation solution  in DPBS 10X and Milli-Q water) at RT for 20 min and permeabilized with 1X Perm/wash (BD) buffer at RT for 10 min. Staining with metal-labeled intracellular Abs and an iridium nucleic acid intercalator in 1X Perm/Wash was carried out as for extracellular staining, but without a new permeabilization step. Cells were stored overnight with 0.1 μM iridium nucleic acid intercalator in fixation mixture. The following day, cells were washed with Milli-Q water, resuspended in 1 ml Milli-Q water and filtered using a 35-μm nylon mesh cell strainer (BD Biosciences), before the addition of EQ Four-Element Calibration Beads , according to the manufacturer's instructions. Acquisition of each sample was performed two times in succession on a CyTOF or Helios instrument (Fluidigm). No autosampler was used for sample acquisition. Moreover, the dual sample loop system was used for sample acquisition performed on the CyTOF. CyTOF settings were parametrized following the quality control of the instrument.For each sample, 3 × 10Data were acquired using EQ™ Four-Element Calibration Beads, normalized using Rachel Finck's MATLAB normalizer, and concatenated using the FCS file concatenation tool . Cytometric profiles were manually gated to exclude the EQ™ Four-Element Calibration Beads, select singlets, and gate out non-specific background generated by metal-conjugated Ab binding to eosinophils , as repracceptable and non-ambiguous nearest neighbors. In details, each time two cells from different cytometric profiles are identified as acceptable and non-ambiguous nearest neighbors, CytoBackBone merges them and a new cell with a combined phenotype is created. This new cell is added to the resulting cytometric profile and its phenotype corresponds to the average marker expression for the set of common markers. For the non-common markers , the phenotype of this cell corresponds to the specific marker expression. CytoBackBone is implemented in R and the source code can be found at: https://github.com/tchitchek-lab/CytoBackBone. Each merge realized by CytoBackBone was calculated based on the set of common markers between the cytometric profiles to be merged . A distance threshold of 3 was used for all merging.The CytoBackBone algorithm  was usedSPADE analyses were generated using the public version of the SPADE R package. viSNE representations were generated based on the Barnes-Hut implementation of the t-SNE algorithm. Dot-plot visualizations were performed using Cytobank software . SPADE analyses were set up and carried out with a down-sampling parameter of 5%. A random pre-downsampling was used to select 67,000 cells from each sample  before the SPADE analysis. Full upsampling was eventually performed after the cell cluster identification by SPADE. Categorical marker heatmaps were generated using the SPADEVizR package . Marker To characterize the phenotypes of leukocytes obtained from HIV-infected patients and healthy donors, we used three different mass cytometry panels. Phenotypic information from the different cytometry profiles was combined using the CytoBackBone algorithm. Such multidimensional phenotype characterization was performed based on the simultaneous expression of 72 markers.We collected whole blood from three non-viremic HIV-infected patients on combined antiretroviral therapies (named PAT-1 to PAT-3) and three healthy donors (named HEA-1 to HEA-3), as indicated in For each patient, phenotypic information from various mass cytometry panels was merged using the CytoBackBone algorithm. Thus, for each of the six subjects, we generated a merged cytometric profile of 72 cell markers.For each cytometric profile, the number of cells obtained before and after merging is shown in The SPADE algorithm was then used to identify 500 cell populations in the whole dataset of the six merged cytometric profiles. The 72 markers were used as clustering markers to capture the maximal cell diversity . For eacWe then generated a categorical heatmap representing the relative marker expression for each cluster to interpret the cell-cluster phenotypes . T cell,The 500 identified clusters were classified into two categories according to their cell abundance. The first category, named Enrichment Trend Clusters (ETCs), corresponded to clusters for which the cell abundance was specific to either HIV patients or healthy donors (cell abundance fold-change >4). The second category corresponded to other cell clusters (clusters without a specific enrichment trend). The trends of cell-cluster abundance are displayed at the bottom of the heatmap . The sumThe localization of the ETCs in the heatmap revealed groups of T cell, B cell, PMN, NK cell, and monocyte clusters that were homogeneously enriched either in HIV-infected patients or healthy donors. ETCs specific to the HIV condition were almost exclusively composed of cells from HIV-infected patients, whereas the number of cells from healthy subjects was negligible. Inversely, the enriched clusters for the healthy (HEA) condition were almost exclusively composed of cells from healthy donors, whereas the number of cells from HIV-infected patients was negligible. We identified two pDC clusters: one enriched in the HEA condition, and another in the HIV condition. We compared the phenotypes of CD4 T cells, CD8 T cells, B cells, PMNs, NK cells, monocytes, and pDCs of HIV-infected patients and healthy donors using this heatmap representing the level of 72 cell markers. No specific groups of basophils or mDCs were associated with a specific individual group. The cell clusters of these populations were thus annotated on the SPADE tree and the cells directly analyzed using biplot representations . The Cytmid, CD11alow, CD11bneg, CD27low, CD38neg, CD83neg, Granzyme Aneg, and Granzyme Bneg/low. In contrast, CD8 T cells from HIV-infected patients were CXCR4high, CD11ahigh, CD11bmid, CD27mid, CD38mid, CD83mid, Granzyme Ahigh, and Granzyme Bhigh or CXCR4high, CD11amid, CD11bmid, CD27high, CD38mid, CD83mid, Granzyme Amid, and Granzyme Bmid. Although low in abundance, we observed a CD32+ subpopulation in HIV-infected patients. Additionally, CD8 T cells from HIV-infected and healthy individuals were mainly CCR7neg, although a set was CCR7high in healthy subjects.Most CD8 T cells from healthy subjects were CXCR4low, CD27low, CD38neg, CD83neg, and Granzyme Bneg/low. In contrast, CD4 T cells from HIV-infected patients were CD11amid, CD27mid, CD38mid, CD83mid, and Granzyme Bmid. Although low in abundance, we observed a CD32+ subpopulation in HIV-infected patients.CD4 T cells from healthy subjects were CD11ahigh, HLADRbright, CD38mid, and CD83high, whereas B cells from healthy individuals were CXCR4mid, HLADRmid/high, CD38neg, and CD83neg.B cells from HIV-infected patients had higher expression of CXCR4, HLADR, CD38, and CD83 than healthy subjects. Indeed, B cells from HIV-infected patients were CXCR4mid, CD11alow, CD11bneg, CD38neg, Granzyme Aneg, Granzyme Bneg, and Perforinneg, whereas they were CXCR4high, CD11ahigh, CD11bmid, CD38high, Granzyme Ahigh, Granzyme Bhigh, and Perforinhigh in HIV-infected patients.NK cells from healthy donors were CXCR4high, CD11bhigh, CD32high, CD38high, CD64high, and TLR2mid. However, PMNs from healthy donors were CD11alow, CD11blow, CD32neg, CD38neg, CD64neg, and TLR2low. PMNs from healthy donors also exhibited a set of CCR7high clusters. Basophils from HIV patients were CD11bhigh, CD38mid, CD62Lhigh, whereas they were CD11bneg, CD38neg, and CD62Lneg in healthy donors.PMNs from HIV-infected patients were CD11amid, CD11bneg, CD11cmid, CD32neg, CD38neg, TLR2mid, and MCP1neg, whereas they were CD11ahigh, CD11bhigh, CD11chigh, CD32high, CD38high, TLR2high, and MCP1mid/high in HIV-infected patients. Although CD64, CD83, CD86, and HLADR were highly expressed by both HIV-infected patients and healthy donors, their levels were higher in HIV-infected patients.Monocytes from healthy donors were CD11amid, CD11bmid, CD83bright, CD86bright, TLR2high, and HLADRbright, whereas those from healthy donors were CD11alow, CD11bneg, CD83mid, CD86mid, TLR2mid, and HLADRhigh.The mDC population can be split into two parts based on the expression of HLADR. However, the modifications of protein expression levels in HIV-infected patients were similar for the two mDC populations. mDCs from HIV-infected patients were CD11amid and Granzyme Bmid, unlike pDCs from healthy donors which were CD11bneg and Granzyme Bneg. Although HLADR was highly expressed in both HIV-infected patients and healthy donors, its level was higher in HIV-infected individuals.pDCs from HIV-infected patients were CD11bHere, we have described HIV infection-dependent phenotypic changes in T cells, B cells, NK cells, PMNs, monocytes, dendritic cells, and basophils. Overall, the monocyte and PMN populations appeared to be the cell populations most highly affected by HIV infection.We next intended to confirm several phenotypic changes obtained with the combined cytometry profiles of 72 markers. To do so, we designed a single mass cytometry panel of 25 cell markers targeting the monocyte population, which was used to stain six new healthy donors (named HEA-4 to HEA-9), three previously used HIV-ART patients (named PAT-1 to PAT-3) and three new HIV-ART patients (named PAT-4 to PAT-6).The monocyte population was used for validation based on two main reasons. First, monocytes were the most impacted population by HIV and were thus of strong relevance in term of disease characterization. Second, we previously predicted the upregulation of CD11b, CD32, CD64, CD83, CD86, and TLR2 in monocytes from HIV-infected patients. However, these markers were located on different panels. CD32, CD64, and CD86 were present in the backbone, whereas TLR2 was present in panel #A, CD11b in panel #B, and CD83 in panel #C . TherefoLeukocytes from the original three HIV-infected patients (named PAT-1 to PAT-3), three new HIV-infected patients (named PAT-4 to PAT-6), and six new healthy donors (named HEA-4 to HEA-9)  were stap = 0.0011), CD32 (p ≤ 0.0001), CD64 (p = 0.0028), CD83 (p = 0.0016), CD86 (p = 0.0410), and TLR2 (p = 0.0008)  . In addiResults from the validation experiment were compared to those obtained from the combined mass cytometry profiles using classical dot plots . We obseSo far, multi-tube cytometry approaches have not been used in the context of chronic inflammation characterization. In addition to the technical difficulties to perform these experiments using mass cytometry panels, the analysis of such complex data requires further bioinformatics approaches. Here, we addressed these analysis challenges using the SPADE and SPADEVizR pipelines. Even if this study is based on a restricted number of samples from HIV-infected patients, the methodology that we describe here demonstrates that multi-tube cytometry can be applied for characterizing complex diseases.Here, we characterized the HIV inflammation at an unprocessed phenotypical resolution of 72 cell marker. We detected the upregulation of several proteins in HIV-infected patients. Among them, CD11a and CD11b were upregulated in PMNs, monocytes, mDCs, NK cells, and T cells. CD11b was also upregulated on pDCs. Other upregulated proteins included: CD38 on PMNs, monocytes, NK cells, basophils, B cells, and T cells; CD83 on monocytes, mDCs, B cells, and T cells; and TLR2, CD32, and CD64 on PMNs and monocytes. The monocyte and PMN population appeared to be strongly affected by the HIV infection, as CD11a, CD11b, CD32, CD38, CD64, CD83, CD86, and TLR2 were upregulated in these populations. Overall, these results show that HIV infection induced a specific environment that similarly affected various immune cells in these patients.Following the detection of Pathogen Associated Molecular Patterns (PAMP) in tissues by cell populations expressing pattern recognition receptors (PRRs), different cytokines and chemokines are produced. The release of these inflammatory proteins leads to the recruitment of innate immune cells, such as neutrophils, monocytes, dendritic cells, and NK cells , 26. WheThe HIV infection induces an exhaustion of different innate immune cells, such as neutrophils, monocytes, and dendritic cells. Indeed, we demonstrated that the production of IL-1α, IL-6, and TNF-α were delayed following different TLR stimulations in HIV-infected patients . These p+ T cells loss, and a high HIV viral load  but are less effective than monocytes and NK cells. Finally, neutrophils and monocytes can perform antibody-dependent cellular phagocytosis of infected cells and immune complexes . Thus, pCD32 and CD64 also mediate ADCC, which is important in the response against HIV. ADCC is mostly performed by NK cells, but we did not observe an upregulation of Fc receptors on this population. However, monocytes and PMNs also carry out ADCC –37. The We also detected an overexpression of MCP-1 on monocytes. MCP-1 is a chemokine that attracts monocytes, lymphocytes, and polymorphonuclear cells. The HIV-NEF protein is known to upregulate the expression of MCP-1 in astrocytes, the most abundant cell type in the brain . The detAlterations in cytokine expression profiles have reported in treated and non-treated HIV-infected individuals. Here, several cytokines were included in our antibody panels, such as IL-1α, IL-1β, IL-1RA, IL2, IL4, IL6, IL8, IL10, IL12, IP-10, IFN-α, IFN-γ, MIP-1β, TNF-α, and MCP-1. Except for MCP-1, no signal of these cytokines was detected in the different leukocyte populations. This no detection of cytokines is explained by the fact that leukocytes were not stimulated by ligands such as LPS or R848. Without such stimulation, the expression level of cytokines inside leukocytes is too low to be detected.low and CD16high) were found in treated HIV-infected patients, neither before nor after the cell merging process. Indeed, all monocytes identified in this study were CD14high and CD16neg/low. However, we found that all monocytes up-regulated the expression of CD11a, CD11b, CD11c, CD32, CD38, TLR2, and MCP-1. Thus, these results suggested that both classical and non-classical monocytes were impacted.In our study, no non-classical monocytes  enhance the spread of HIV by activating TLR2-dependent signaling . Indeed, nearly 90% of HIV-infected patients are CMV positive contrasting with about 60% in healthy donors.Because HIV-1 infected patients were ART-treated, we suppose that the replication of HIV is not involved in the reported alterations. However, the virus could still play a role, as increasing pieces of evidence point to the persistence of HIV in tissues despite undetectable viral load in plasma , 61. HerAlthough our combined cytometry panel is composed of 72 cells markers, several important markers for phenotypical annotation of cell subsets are unfortunately absent. Indeed, markers such as CD10, CXCL13, CD278 (follicular T cells), NKG2A, NKp44 (NK cells), T-bet, GATA, RORγt, IL-17 (ILC), or FoxP3 (T regs) were not included in our antibodies panels. Thus, further research must be conducted to extend this approach to characterize all cutting-edge subpopulations involved in HIV.bright and TLR2neg in the single-tube experiment. However, we did not observe this result in the heatmap from the multi-tube 72-marker experiment. The multi-tube 72-marker experiment was performed with three HIV-ART patients (named PAT-1 to PAT-3), whereas the single-tube experiment was performed with six HIV-ART patients . After deeper analysis, we found this CD64bright and TLR2neg monocyte population to be detectable in patients PAT-2, PAT-4, PAT-5, and PAT-6. Thus, it was normal to not observe this population on the 72 cell-marker heatmap, as monocytes from patients PAT-1 to PAT-3 were mostly CD64bright and TLR2high.In the viSNE analysis, monocytes were partly CD643. These results are in agreement with several publications showing persistent chronic immune activation in patients on prolonged ART for whom increased levels of soluble biomarkers (such as sCD14 and IP10) were detected  Ile de France VII, under protocol number PP 14-003. All subjects gave written informed consent to participate in this study.AL, NT, and AC: conceptualization and methodology, investigation. AL, NT, AC, RL, and OL: validation and writing—review and editing. AL and NT: formal analysis and writing—original draft. AL and OL: resources. AC and RL: funding acquisition. AC, RL, and OL: supervision.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.


Triple-negative breast cancer (TNBC) is cancer that tested as negative for estrogen receptors (ER), progesterone receptors (PR), and excess human epidermal growth factor receptor 2 (HER2) protein which accounts for 15%–20% of all breast cancer cases. TNBC is considered to be a poorer prognosis than other types of breast cancer, mainly because it involves more aggressive phenotypes that are similar to stem cell–like cancer cells . Thus, targeted treatment of TNBC remains a major challenge in clinical practice. This review article surveys the latest evidence concerning the role of genomic alteration in current TNBC treatment responses, current clinical trials and potential targeting sites, CSC and drug resistance, and potential strategies targeting CSCs in TNBC. Furthermore, the role of insulin-like growth factor 1 receptor (IGF-1R) and nicotinic acetylcholine receptors (nAChR) in stemness expression, chemoresistance, and metastasis in TNBC and their relevance to potential treatments are also discussed and highlighted. Breast cancer is the most frequently diagnosed malignancy and the main cause of cancer-related death in women. Globally, approximately 2.1 million new female breast cancer cases and 627,000 deaths were estimated to have occurred in 2018 [So-called triple negative breast cancer (TNBC) is more commonly diagnosed in women younger than 40 years compared to hormone-positive breast cancer . The hisTP53) and several DNA repair genes, particularly the BRCA genes, are either mutated or abnormally expressed in TNBC. These molecular characteristics may influence chemotherapy sensitivity to direct DNA-damaging agents such as platinum [TNBC is clinically heterogeneous, with deviations in morphology, mutational phenotype, and signaling profiles between tumors. Notably, the diagnostic criteria have not been developed to distinguish a distinct biologic subtype of breast cancer—a “triple-negative” phenotype. A histologic subtype, medullary carcinoma, despite generally being triple-negative, has a very good prognosis . Due to platinum .+, EGFR−, and c-KIT− subgroups [In 2000, Perou et al. discovered a classification of breast cancer based on gene expression patterns. The triple-negative clinical phenotype mostly comprises the basal cell–like subgroup . Howeverubgroups .β (TGF-β) and receptor type III (TGFBRIII) were demonstrated to drive migration and invasion of MSL cell lines both in vitro and in vivo [During the last decade, numerous studies have developed exclusive molecular classifications for TNBC. Rody et al. first distinguished a molecular subgroup by defining 16 metagenes within the group . In addi in vivo . These sp < 0.001). Thus, a confirmatory biopsy of a suspected lesion should be obtained when possible [Treatment for TNBC remains a major clinical challenge due to the lack of causally proven oncogenic drivers through which to target the vast disease heterogeneity ,23. To dpossible .Because patients with TNBC commonly do not achieve a pCR following chemotherapy, the selection of chemotherapy to use against different TNBC subtypes is being debated . NeoadjuBRCA1 expression, caused by promoter methylation and p53 mutations, conferred good prognosis to cisplatin treatment [CD73 expression has been associated with doxorubicin resistance in patients with TNBC [p53 and PIK3CA, loss of PTEN and RB1, amplification of cyclin E1, and increased expression of MYC and HIF1-α [CDK4, MCL1, JAK2, AKT1, and EGFR) and loss of mutations in BRCA1/2, ATM, and CDKN2A [Several studies, including in vitro and in vivo studies and clinical trials, have defined genomic effects inherent to TNBC response to treatment. Silver et al. demonstrated that the alteration of reatment . Similarith TNBC . Anotherd HIF1-α . Furtherd CDKN2A . These fA summary of current trials of single-agent treatments or combinations of different target therapeutic reagents and chemotherapy is provided in PARP1 could induce double-strand DNA breaks, where DNA damage is repaired through a BRCA pathway–dependent homologous recombination mechanism under normal circumstances. Therefore, inhibition of PARP is particularly useful in BRCA-mutated breast cancer with aberrant DNA damage responses (DDR) [BRCA-mutated breast cancer and TNBC, the efficacy and safety of PARP inhibition is being tested in both settings. Among the subset of 121 BRCA mutation carriers with metastatic triple-negative disease in the OlympiAD trial has been grouped to anthracycline and a taxane in either the adjuvant or metastatic setting. Those that were randomly assigned to olaparib exhibited improved PFS relative to those receiving chemotherapy  [Inhibition of es (DDR) ,32,33. G29–0.63) ,35. Nota29–0.63) ,40,41,42The epidermal growth factor receptor (EGFR/HER1) may be the most well-known protein that is overexpressed in cancers, and for which several monoclonal antibodies and small-molecule inhibitors exist. Several phase II clinical trials have assessed the efficacy of cetuximab, an anti-EGFR monoclonal antibody, which has exhibited modest activity in combination with chemotherapy against advanced TNBC ,44. In aBoth normal and malignant breast tissue express the androgen receptor (AR) . LuminalImmunologic escape describes the stage at which malignant clones acquire the capability of evading the adaptive immune system. Programmed cell death 1 (PD-1) is a transmembrane protein expressed on T cells, B cells, and NK cells: PD-1 ligand (PD-L1) binds to PD-1 and then directly inhibits apoptosis of the tumor cell while promoting peripheral T effector cell exhaustion and conversion of T effector cells to Treg cells . In addiIn recent decades, the theory of CSCs or cancer stemness has captured much attention and interest. It is thought that this theory has potential to revolutionize our understanding of the cellular and molecular events during cancer progression and how they contribute to drug resistance, tumor recurrence, and metastasis . Among sIn addition, another cell type, quiescent non-CSCs, also resists anti–mitotic drugs. In primary tumor, growth phases transition between proliferation and dormancy in non-CSCs are determined by genetic and epigenetic alternations ,65. DrugThe concept of cancer stemness or CSCs is currently used to explain the mechanism of MDR. These cells are recognized as a group of cells that possess much higher endogenous resistance against radiation and chemotherapy than non-CSC differentiated tumor cells . CSCs an+CD24−/low and ALDH1 positivity [+/CD24−/low/ALDH+ BCSC-like subset of the TNBC cell line [The study of CD44/CD24 and ALDH1 expression could be the most appropriate to identify BCSCs with distinct levels of differentiation from breast cancer populations. In an analysis of 466 invasive breast carcinomas and eight breast cancer cell lines, basal-like breast cancer harbored the highest percentage of tumor cells with the CSC phenotype CD44sitivity . Clinicasitivity ,76. Enrisitivity ,78,79. Fell line . In patiell line ,26,27.CSCs are located in a specialized microenvironment termed the niche that is mainly composed of cancer-associated fibroblasts (CAF), endothelial cells (ECs), mesenchymal SCs (MSC), tumor-associated macrophages (TAM), and extracellular matrix (ECM), which play different roles in orchestrating therapy resistance ,82,83. SABCB1), the multidrug resistance protein 1 , and the breast cancer resistance protein , have been implicated as major efflux transporters responsible for multidrug resistance in cancer cells [ABCG2 mRNA expression and response in a subgroup of patients receiving anthracycline-based chemotherapy , and such a correlation did not exist in the cyclophosphamide, methotrexate, and 5-fluorouracil-treated group of patients [ABCB1 genes in five paclitaxel-resistant TNBC cell lines . Furthermore, a link between ABCB1 and the Hedgehog pathway supports the relationship between CSCs and ABC transporters [The ATP-binding cassette (ABC) transporters belong to a family of 49 membrane proteins usually related to the efflux of small molecules and compounds from the cytosol to the extracellular medium using ATP hydrolysis. Due to their ability to expel toxic chemicals, they participate in the development of MDR ,98. Manyer cells . Many ABer cells . Thus, ser cells ,102,103.er cells . In breapatients . A searcsporters .The mechanisms responsible for EMT and CSC-related drug resistance remain uncertain in most cases. One hypothesis suggests that cells undergoing EMT enter into a quiescent state and no longer divide ; howeverα (HIF-1α) is the main regulator of cellular responses to hypoxia. It is ubiquitinated at high oxygen levels and subsequently degraded. At decreased oxygen levels, HIF-1α is activated upon ubiquitination inhibition, translocates into the nucleus, dimerizes with HIF-1β, and activates the transcription of specific genes [α has long been recognized as a pivotal orchestrator of cancer cell response to hypoxic microenvironments by regulating the expression of over 60 genes involved in metabolic reprogramming and pH balance, cell proliferation/survival, apoptosis, angiogenesis, SC maintenance, matrix remodeling, metastasis, and resistance to radiotherapy and chemotherapy [α–dependent manner [α overexpression identified by immunohistochemistry of tumor biopsies has been associated with increased mortality [α expression has been reported to be correlated with MDR in breast cancers. For example, expression can be induced by treatment of breast cancer cells with doxorubicin under normoxic environments [α, the expression of EMT and stemness activators such as WNT, Hedgehog, and NOTCH pathways, as well as other stemness markers such as FOXA2, cMET, CD133, NANOG, SOX2, SOX17, and PDX1 occurs [α signaling pathway decreases the production of ROS and preserves SC properties leading to drug resistance in CSCs [The hypoxic niche plays an important role in the maintenance of SC characteristics during embryonic development and self-renewal ,110. In ic genes . HIF-1α otherapy ,114. Expt manner ,116, whit manner . Clinicaortality . Increasortality . Furtherronments . Furtherronments . Through1 occurs ,123. Bec1 occurs ,125. Und1 occurs ,127, whe in CSCs ,128. In  in CSCs ,130.p = 0.003). The pCR rate and ALDH1 expression were significantly correlated in patients with TNBC (p = 0.003). ALDH1(+) breast cancers tend to be aggressive with poor prognoses [Aldehyde dehydrogenase (ALDH) is another molecule responsible for ROS decrease and is also a CSC marker . It comprognoses .+CD24–/low BCSCs, DNA single-strand break repair is particularly active and linked to APE1 upregulation [+CD24–/low BCSCs by epigenetically repressing DNA repair [2-M checkpoint to survive radiotherapy and eventual metastatic relapse. This result was further established following the inhibition of CHK1, which resensitized the cells [CSCs are multipotent SCs responsible for the long-term clonal maintenance and growth of most human neoplasms. They can coexist in a cycling and quiescent state as they acquire mutations that further increase heterogeneity . CSCs ofgulation . In addigulation . The mosgulation . Comparegulation . EZH2 paA repair . The bashe cells . Furtherhe cells . CollectSeveral clinical trials have been designed that combine conventional chemotherapy and adjuvant therapies in the hope of eliminating both actively dividing cells and CSCs. These CSC-targeted adjuvant regimens may either attack stem-related pathways or enhance the drug sensitivity of CSCs in distinguished ways . In the + cells for the selective killing of BCSCs using conventional chemotherapy [Previous studies have revealed that increased cyclophosphamide sensitivity of lung cancer cells could be achieved by knockdown of two ALDH isoforms . As prevotherapy .Due to limited specific markers, several studies have tried to eliminate CSCs by targeting highly activated signaling pathways in these cells. A number of signaling pathways have been associated with the therapy-resistant phenotype of BCSCs, including Notch, Hedgehog, and Wnt, which regulate apoptosis escape, maintenance of a stem cell niche, and increased invasion capacity ,149,150.0 cell cycle arrest at the single-cell level, which resembles CSCs in primary tumors [0/G1 phase to the S-phase of the cell cycle, induce reversible or irreversible G0/G1 cell cycle arrest in various cancer models [β2 [Cancer cell dormancy is attributable to Gy tumors . The proy tumors . To datey tumors . The cony tumors ,157. Simy tumors . Inhibitr models ,160. Curdels [β2 ,162,163,dels [β2 . An awakdels [β2 . Similardels [β2 . In addidels [β2 . Similardels [β2 . Lastly,dels [β2 . In addidels [β2 . Interesdels [β2 ,171,172.dels [β2 . In summThe Warburg effect of aerobic glycolysis describes rapidly proliferating cancer cells that overtake angiogenesis, resulting in areas of low oxygen that accelerate glycolytic production of ATP . However+ BCSCs tumorigenicity through EGFR/STAT3 signaling in a mouse model [Autophagy homeostasis has been strongly associated with CSC physiology, such as tumorigenesis, differentiation, plasticity, migration/invasion, and pharmacological, viral and immune-resistance . Currentse model . Furtherse model , chromosse model , cytokinse model , and SC se model  in a varse model . For exase model ,208. Simse model . In summet al. investigated the use of metformin target KLF5 for degrading and preventing the activation of KLF5 downstream target genes NANOG and FGF-BP1, which consequently reduced the subpopulation of BCSCs in TNBC [Metformin, the most well-known medication for type II diabetes, is a candidate for reducing CSC resistance ,211. It  in TNBC . Current in TNBC . Doxycyc in TNBC ,216. Dox in TNBC . CurrentThe CSC niche regulates intrinsic resistance . Therefoβ suppresses the CSC properties, resulting in decreased tumor sphere formation, EMT expression, and migration, thereby promoting an epithelial phenotype with reexpression of CD24 [Evidence has shown chronic inflammation serves as a protumorigenic factor. High serum levels of IL6, an inflammatory cytokine, can predict the development of hepatitis B virus (HBV)-infected hepatocellular carcinoma (HBV-HCC) . The nic of CD24 . Studies of CD24 , and theα7-nAChR/YAP1/E2F1 axis in non-small-cell lung cancer (NSCLCs). Tumor cells acquire stemness functions to initiate self-renewal proliferation and migration in NSCLCs [Nicotine is a component of tobacco and e-cigarettes, and its receptors  are carcinogens contributing to various cancers. A previous study demonstrated that nicotine not only results in the upregulation of the stemness-related genes OCT4, SOX9, HES1, ALDH1, and STAT3 in pancreatic tumors but also promotes EMT behavior and metastasis . In addin NSCLCs . These rα [The IGF signaling system controls energy metabolism, stem cell self-renewal, cell growth, and body size in normal physiology ,233,234.α ,224. Morα ,242,243.α , and actα . Chang eα . Activatα . Furtherα ,223. Theα7-nAChR subtype is associated with lung [α9-nAChR, and STAT3 regulated the enrichment of side population cells with CSC-like properties [α9-nAChR across human cancers [α9-nAChR, acts as an antimetastasis agent in nicotine-induced breast cancer. In summary, these results indicate the crucial role of α9-nAChR in the identification of biomarkers and therapeutic targets and agents.Numerous studies have identified the distinguished nAChRs subtypes as mediating tobacco-related cancer development and progression. The nAChRs transduce various signaling cascades upon activation, which govern several pathological conditions, namely tumorigenesis ,254,255,ith lung ,255, blaith lung ,261, colith lung ,268,269,ith lung ,259, andith lung ,262,264 ith lung . Studiesith lung ,270,271.operties . Therefooperties ,266. Mos cancers . In addiIn addition to molecular approach, there are several functional profiling approaches to establish precision treatment strategies against TNBC. Heiser et al. have studied that the variant responses to 77 therapeutic compounds are occurred across numerous breast cancer cell lines including TNBC subtype, and approximately one third showing these specific responses depend on subtype, pathway, and/or genomic aberration . Since lIn this review, we pointed out how multiple mechanisms underlying CSC maintenance give rise to not only tumor survival but also plasticity to drug resistance, particularly under unfavorable stress . Currentα9-nAChR in TNBCs (The considerable disease heterogeneity, both intertumor and intratumor, remains the major obstacle to identifying actionable targets in TNBC. Due to the lack of well-defined molecular targets, current treatment options for TNBC commonly focus on cytotoxic chemotherapy. Despite different TNBC subtypes presenting distinct treatment responses, subtype-based chemo regimen selection remains debatable. In addition, cytotoxic chemotherapy against actively dividing cells within a tumor is not capable of eliminating dormant CSCs, which results in low pCR and short DFS in patients with TNBC. Although more aggressive chemotherapy has resulted in improved prognosis, it usually leads to serious deterioration in quality of life. This has prompted endeavors to develop novel drugs and identify effective subtype-specific therapeutic strategies, such as anti-IGF-1R and anti-in TNBCs . α9-nAChR are reasonable strategies to combine with chemotherapies to attack the CSCs of TNBCs (Several studies over a number of years have examined target therapies against CSCs in hopes of eradicating MDR. However, only targeting these CSCs does not succeed in tumor eradication either. While “one size does not fit all TNBCs” is the lesson learned from the past, the same rule applies in so far as the previous failure in CSC-targeted therapy does not determine all cases. Recent studies have demonstrated satisfying results when they have repositioned a CSC-targeted regimen in other breast cancer prescriptions, such as in combination with chemotherapy or time-sequential treatment rather than co-treatment. Moreover, anti-IGF-1R or anti-of TNBCs . Given t


Oral cancer is a major public health burden worldwide. The lack of biomarkers for early diagnosis has increased the difficulty in managing this disease. Recent studies have reported that neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, is upregulated in various tumors. In our study, we found that NGAL was significantly downregulated in primary malignant and metastatic tissues of oral cancer in comparison to normal tissues. The downregulation of NGAL was strongly correlated with both degree of differentiation and stage (I–IV); it can also serve as a prognostic biomarker for oral cancer. Additionally, tobacco carcinogens were found to be involved in the downregulation of NGAL. Mechanistic studies revealed that knockdown of NGAL increased oral cancer cell proliferation, survival, and migration; it also induced resistance against cisplatin. Silencing of NGAL activated mammalian target of rapamycin (mTOR)signaling and reduced autophagy by the liver kinase B1 (LKB1)-activated protein kinase (AMPK)-p53-Redd1 signaling axis. Moreover, cyclin-D1, Bcl-2, and matrix metalloproteinase-9 (MMP-9) were upregulated, and caspase-9 was downregulated, suggesting that silencing of NGAL increases oral cancer cell proliferation, survival, and migration. Thus, from our study, it is evident that downregulation of NGAL activates the mTOR pathway and helps in the progression of oral cancer. Despite significant advancements in the management of oral cancer, it is one of the prime concern worldwide, accounting for approximately 128,000 deaths annually . RegardlOver the past two decades, neutrophil gelatinase-associated lipocalin (NGAL) has received enormous attention in the clinic as a biomarker of kidney injury, cardiovascular injuries, and cancer ,5,6,7,8.In the present study, we have examined the expression of NGAL in different stages, grades, tumours from different tissues, degree of differentiation, and different processes of development of oral cancer. We found that NGAL plays a pivotal role in different processes of oral cancer development such as survival, proliferation, invasion, migration, and resistance to chemotherapeutic agents.Tissue microarray (TMA) slides for head and neck squamous cell carcinoma (Cat no: HN803b) and oral squamous cell carcinoma (Cat no: OR802) were purchased from US Biomax, Derwood, MA USA.Expression of NGAL was determined by immunohistochemical analysis. Histostain plus kit  was used according to the manufacturer’s protocol. Anti-hNGAL monoclonal antibody was purchased from . The TMAs were deparaffinised and rehydrated using xylene and ethanol and were blocked with 3% hydrogen peroxide in methanol for 30 min. After the antigen retrieval, the sections were incubated in blocking solution for 30 min and then were incubated with primary antibody (1:10 dilution) at 4 °C overnight. The following day, the sections were incubated with secondary antibody for 1 h at room temperature, stained with DAB counter stained with haematoxylin, and were mounted using DPX.All slides were observed under a Nikon Eclipse Ti-E microscope, and the intensity of immunoreactivity for NGAL was examined. The staining intensity was graded on a scale of 0 to 3+ . The percentage of cells positive for NGAL were graded by the following protocol: grade 0 intensity (<10% positive cells); grade 1+ intensity (10–25% positive cells), grade 2+ intensity (25–50%), grade 3 intensity (50–75% positive cells), and grade 4 intensity (75–100% positive cells). The staining intensity score and the percent immunoreactivity score were then multiplied to obtain a composite score.N′-Nitrosonornicotine , 4-Nitroquinoline N-oxide , Cisplatin (PHR1624), and 5-Flurouracil (5-FU) (F6627) were purchased from Sigma-Aldrich, Saint Louis, MO, USA.4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone , ®, Grand Island, NY, USA) and 1× Penstrep . The cells were cultured and maintained at 37 °C in 5% CO2 and 95% humidity.Human squamous cell carcinoma of the tongue, SAS cells were procured from Rajiv Gandhi Centre for Biotechnology (RGCB), Trivandrum, India. These cells were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , Phospho-S6 Ribosomal protein (Ser235/236) , LC3B , Caspase-9 , Bcl-2 , MMP-9 , and cyclin D1 , GAPDH  were purchased from Cell Signaling Technology, Danvers, MA, USA. Antibodies against NGAL , anti-mouse secondary antibody , and anti-rabbit secondary antibody  were purchased from Abcam.4 cells/well in 1 mL of medium in a 24-well plate. The next day, cells were transfected with shRNA control and shNGAL plasmids (2 µg of DNA) using X-treme gene 9 DNA transfection reagent  for 48 h. The medium containing transfection reagent was replaced with fresh DMEM medium and the cells were allowed to recover for 24 h. Then SAS cells were selected with 1μg/mL puromycin and knockdown clones were established.shRNA-mediated knockdown of NGAL was carried out in the SAS cell line. Human shNGAL plasmids  and puro3 cells/well were seeded in 96-well plates in sextuplicate and incubated for 24 h and 48 h time points. After each time point, 10 µL of 3- 2,5-diphenyl tetrazolium bromide (MTT)  was added to the cells and was further incubated for 2 h at 37 °C. The MTT solution was removed and 100 μL of DMSO  was added to each well and the absorbance was then measured at 570 nm using an Infinite M200 Pro  after 1 h.Briefly, 2 × 105 cells/well, and, after 24 h, cells were trypsinized, washed with phosphate-buffered saline (PBS), and fixed with 75% ethanol at −20 °C overnight. The following day, cells were washed with PBS, treated with PI/RNase solution  for 20 min in the dark and analyzed using a flow cytometer . 25,000 cells in each sample were analyzed to obtain a measurable signal. The data were analyzed on FCS express6 .Control shRNA and shNGAL cells were plated at a density of 1 × 103 cells/well. The cells were grown for fifteen days, then colonies were fixed with 70% ethanol and were stained with crystal violet. Pictures of individual wells were taken and were analyzed using imageJ 1.x software pyrene, nicotine, NNK, NNN, dibenzo[a]pyrene, benzene, nitrobenzene, 2-toluidine, and 2-6-dimethylaniline. Upon activation, NNK and NNN induce mutations in tumour suppressor genes and oncogenes; they form DNA adducts that result in tumour initiation ,35,36,37Next, we found that downregulation of NGAL induced oral cancer cell proliferation, survival, invasion, and migration. Many studies report that NGAL plays a key role in the invasion and migration of oral cancer and other cancers. Recently, Lin et al., 2016 reported that knockdown of NGAL increased in vitro cell motility and in vivo metastases . HoweverFurthermore, AMPK activates p53 during metabolic stress by phosphorylating MDMX on serine 34, resulting in inhibition of p53 ubiquitylation . MoreoveOur results suggest that NGAL is downregulated in oral cancer tissues and is strongly associated with degree of differentiation, stage of the tumour, and lymph node metastases. The tobacco components, primarily NNK, NNN, and the synthetic carcinogen 4-NQO, were implicated in the downregulation of NGAL. Mechanistic studies revealed that knockdown of NGAL augmented cell survival, invasion, and migration by activating the mTOR pathway and also downregulated autophagy via the LKB1-AMPK-p53-Redd1 signalling axis . This su