The Drosophila brain on cocaine at single-cell resolution.

Whereas the neurological effects of cocaine have been well documented, effects of acute cocaine consumption on genome-wide gene expression across the brain remain largely unexplored. This question cannot be readily addressed in humans but can be approached using the Drosophila melanogaster model, where gene expression in the entire brain can be surveyed at once. Flies exposed to cocaine show impaired locomotor activity, including climbing behavior and startle response (a measure of sensorimotor integration), and increased incidence of seizures and compulsive grooming. To identify specific cell populations that respond to acute cocaine exposure, we analyzed single-cell transcriptional responses in duplicate samples of flies that consumed fixed amounts of sucrose or sucrose supplemented with cocaine, in both sexes. Unsupervised clustering of the transcriptional profiles of a total of 86,224 cells yielded 36 distinct clusters. Annotation of clusters based on gene markers revealed that all major cell types (neuronal and glial) as well as neurotransmitter types from most brain regions were represented. The brain transcriptional responses to cocaine showed profound sexual dimorphism and were considerably more pronounced in males than females. Differential expression analysis within individual clusters indicated cluster-specific responses to cocaine. Clusters corresponding to Kenyon cells of the mushroom bodies and glia showed especially large transcriptional responses following cocaine exposure. Cluster specific coexpression networks and global interaction networks revealed a diverse array of cellular processes affected by acute cocaine exposure. These results provide an atlas of sexually dimorphic cocaine-modulated gene expression in a model brain.

Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

Modulation of the Drosophila transcriptome by developmental exposure to alcohol.

BACKGROUND: Prenatal exposure to ethanol can cause fetal alcohol spectrum disorder (FASD), a prevalent, preventable pediatric disorder. Identifying genetic risk alleles for FASD is challenging since time, dose, and frequency of exposure are often unknown, and manifestations of FASD are diverse and evident long after exposure. Drosophila melanogaster is an excellent model to study the genetic basis of the effects of developmental alcohol exposure since many individuals of the same genotype can be reared under controlled environmental conditions. RESULTS: We used 96 sequenced, wild-derived inbred lines from the Drosophila melanogaster Genetic Reference Panel (DGRP) to profile genome-wide transcript abundances in young adult flies that developed on ethanol-supplemented medium or standard culture medium. We found substantial genetic variation in gene expression in response to ethanol with extensive sexual dimorphism. We constructed sex-specific genetic networks associated with alcohol-dependent modulation of gene expression that include protein-coding genes, Novel Transcribed Regions (NTRs, postulated to encode long non-coding RNAs) and female-specific coordinated regulation of snoRNAs that regulate pseudouridylation of ribosomal RNA. We reared DGRP lines which showed extreme upregulation or downregulation of snoRNA expression during developmental alcohol exposure on standard or ethanol supplemented medium and demonstrated that developmental exposure to ethanol has genotype-specific effects on adult locomotor activity and sleep. CONCLUSIONS: There is significant and sex-specific natural genetic variation in the transcriptional response to developmental exposure to ethanol in Drosophila that comprises networks of genes affecting nervous system development and ethanol metabolism as well as networks of regulatory non-coding RNAs.

Pleiotropic fitness effects of the lncRNA Uhg4 in Drosophila melanogaster.

BACKGROUND: Long noncoding RNAs (lncRNAs) are a diverse class of RNAs that are critical for gene regulation, DNA repair, and splicing, and have been implicated in development, stress response, and cancer. However, the functions of many lncRNAs remain unknown. In Drosophila melanogaster, U snoRNA host gene 4 (Uhg4) encodes an antisense long noncoding RNA that is host to seven small nucleolar RNAs (snoRNAs). Uhg4 is expressed ubiquitously during development and in all adult tissues, with maximal expression in ovaries; however, it has no annotated function(s). RESULTS: We used CRISPR-Cas9 germline gene editing to generate multiple deletions spanning the promoter region and first exon of Uhg4. Females showed arrested egg development and both males and females were sterile. In addition, Uhg4 deletion mutants showed delayed development and decreased viability, and changes in sleep and responses to stress. Whole-genome RNA sequencing of Uhg4 deletion flies and their controls identified co-regulated genes and genetic interaction networks associated with Uhg4. Gene ontology analyses highlighted a broad spectrum of biological processes, including regulation of transcription and translation, morphogenesis, and stress response. CONCLUSION: Uhg4 is a lncRNA essential for reproduction with pleiotropic effects on multiple fitness traits.

Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster.

Large multigene families, such as the insect odorant-binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 reintegration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (transcriptional niches) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.

Classification Methods and Identification of Reniform Nematode Resistance in Known Soybean Cyst Nematode-Resistant Soybean Genotypes.

Plant parasitic nematodes are a major yield-limiting factor of soybean in the United States and Canada. It has been indicated that soybean cyst nematode (SCN; Heterodera glycines Ichinohe) and reniform nematode (RN; Rotylenchulus reniformis Linford and Oliveira) resistance could be genetically related. For many years, fragmentary data have shown this relationship. This report evaluates RN reproduction on 418 plant introductions (PIs) selected from the U.S. Department of Agriculture Soybean Germplasm Collection with reported SCN resistance. The germplasm was divided into two tests of 214 PIs reported as resistant and 204 PIs reported as moderately resistant to SCN. The defining and reporting of RN resistance changed several times in the last 30 years, causing inconsistencies in RN resistance classification among multiple experiments. Comparison of four RN resistance classification methods was performed: (i) ≤10% as compared with the susceptible check, (ii) using normalized reproduction index (RI) values, and using (iii) transformed data log10(x), and (iv) transformed data log10(x + 1) in an optimal univariate k-means clustering analysis. The method of transformed data log10(x) was selected as the most accurate for classification of RN resistance. Among 418 PIs with reported SCN resistance, the log10(x) method grouped 59 PIs (15%) as resistant and 130 PIs (31%) as moderately resistant to RN. Genotyping of a subset of the most resistant PIs to both nematode species revealed their strong correlation with rhg1-a allele. This research identified genotypes with resistance to two nematode species and potential new sources of RN resistance that could be valuable to breeders in developing resistant cultivars.

Effect of Lipid Vehicles on Solubility, Stability, and Topical Permeation of Delta-9-Tetrahydrocannabinol.

Delta-9-tetrahydrocannabinol (THC) is one of the most effective antinociceptive agents used in the treatment of peripheral neuropathy. THC is highly lipophilic and susceptible to thermal and oxidative degradation. Identifying appropriate solvents in which THC is stable as well as adequately solubilized is crucial in developing topical dosage forms. Lipid solvent systems are of utmost utility and relevance for formulating highly lipophilic drugs. Hence, the objective of this project was to screen the solubility of THC in lipidic excipients, monitor THC content in the selected vehicles during stability, and study the influence of these excipients on permeation of THC across skin. The solubility of THC in liquid lipid excipients was in the range of 421 to 500 mg/g. The solubility of THC in solid lipid excipients was in the range of 250 to 750 mg/g. THC in its neat form was poorly stable, but when dissolved in lipid-based excipients, its stability improved significantly. THC in lipid excipients was more stable at 4 ± 3°C compared to samples stored at 25 ± 2°C. The antioxidants (butylated hydroxytoluene and ascorbyl palmitate) used in the excipients further improved the stability of THC. The results demonstrated that the liquid and solid lipid excipients used in the study could solubilize THC freely and mitigate the degradation of THC significantly. The binary combination of lipid excipients enhanced THC skin permeation and retention, demonstrating the potential for topical formulation development of THC.

Development and Validation of HPLC Method for Efinaconazole: Application to Human Nail Permeation Studies.

Efinaconazole is the first azole derivative approved by FDA for the topical treatment of onychomycosis. The objective of present study was to develop and validate HPLC method for estimation of efinaconazole in ex vivo human nail permeation study samples. The chromatographic analysis was performed on a HPLC system equipped with diode array detector. The efinaconazole and internal standard (IS) were extracted from the human nail samples by using the protein precipitation method. The samples were injected on to 5 µm Polar C18 100Å, 4.6 mm × 150 mm column. The mobile phase consisted of 0.01 M potassium dihydrogen phosphate: acetonitrile (36:64) and eluent was monitored at 205 nm. The chromatographic separation of drug and analyte was achieved using isocratic elution at flow rate of 1 mL/min with a total run time of 15 min. The efinaconazole and IS were eluted at 6.4 ± 0.5 and 8.3 ± 0.5 min, respectively. The developed method was validated as per FDA guidelines, and the results met with acceptance criteria. The method developed was specific, and the analyte concentrations were linear at range of 50 to 10000 ng/mL (R2 ≥ 0.9981). The validated HPLC method was applied for quantifying efinaconazole in human nail permeation study samples. The permeation of efinaconazole was increased by twofolds with Labarfac CC (15135.4 ± 2233.9 ng/cm2) compared to formulations containing Transcutol P (6892.0 ± 557.6 ng/cm2) and Labrasol (7266.1 ± 790.6 ng/cm2). The study results demonstrate that developed efinaconazole HPLC method can be employed for formulation evaluation and clinical studies.

Genomic variation in captive deer mouse (Peromyscus maniculatus) populations.

BACKGROUND: Deer mice (genus Peromyscus) are the most common rodents in North America. Despite the availability of reference genomes for some species, a comprehensive database of polymorphisms, especially in those maintained as living stocks and distributed to academic investigators, is missing. In the present study we surveyed two populations of P. maniculatus that are maintained at the Peromyscus Genetic Stock Center (PGSC) for polymorphisms across their 2.5 × 109 bp genome. RESULTS: High density of variation was identified, corresponding to one SNP every 55 bp for the high altitude stock (SM2) or 207 bp for the low altitude stock (BW) using snpEff (v4.3). Indels were detected every 1157 bp for BW or 311 bp for SM2. The average Watterson estimator for the BW and SM2 populations is 248813.70388 and 869071.7671 respectively. Some differences in the distribution of missense, nonsense and silent mutations were identified between the stocks, as well as polymorphisms in genes associated with inflammation (NFATC2), hypoxia (HIF1a) and cholesterol metabolism (INSIG1) and may possess value in modeling pathology. CONCLUSIONS: This genomic resource, in combination with the availability of P. maniculatus from the PGSC, is expected to promote genetic and genomic studies with this animal model.

Doppler-derived renal resistive index helps predict acute kidney injury in patients undergoing living-related liver transplantation.

BACKGROUND: Acute kidney injury (AKI) is commonly associated with increased postoperative morbidity in liver transplant (LT) recipients. The aim of this study was to identify the role of renal resistive index (RRI) in predicting AKI and to study the factors associated with AKI in LT recipients. PATIENTS AND METHODS: We performed a single-center, prospective study, including adult living donor LT recipients at our center between January 2018 and September 2019 with no preoperative renal dysfunction. RRI was calculated on ultrasound doppler once preoperatively, and once daily in the postoperative period through postoperative day (POD) six. Patients were grouped into AKI and non-AKI groups for comparison. RESULTS: Fifty patients were included in the study (mean age, 44 years; 20% females). AKI developed in 25 patients (50%). Both groups were similar in baseline characteristics. RRI of ≥ 0.69 on POD 2 predicted AKI (sensitivity 88%; specificity 92%). RRI on the day before AKI diagnosis (0.71 vs. 0.65) and on the day of diagnosis (0.72 vs. 0.65) were significantly increased relative to preoperative baseline. CONCLUSIONS: Doppler-derived RRI is a rapid, non-invasive, and bedside procedure capable of predicting the occurrence of postoperative AKI in LT recipients.

Experimental investigations for assessing the influence of fly ash on the flow through porous media in Darcy regime.

Hydraulic conductivity plays a vital role in the studies encompassing explorations on flow and porous media. The study investigates the compaction characteristics of a river sand (Beas, Sutlej, and Ghaggar rivers) and fly ash mix in different proportions and evaluates four empirical equations for estimating hydraulic conductivity. Experiments show that an increase in the fly ash content results in a decrease in the maximum dry density (MDD) and an increase in the corresponding optimum moisture content (OMC) of sand-fly ash samples. MDD at optimum fly ash content was achieved at low water content, which resulted in less dry unit weight than that of typical conventional fill. In Beas, Sutlej, and Ghaggar sands the optimum fly ash content up to which the hydraulic conductivity value reduced uniformly was found to be 30, 45, and 40%, respectively. Any further increase in the fly ash content results in a negligible decrease in hydraulic conductivity value. The observed hydraulic conductivity of sand-fly ash mix lies in the range of silts, which emboldens the use of sand-fly ash mix as embankment material. Further, the evaluation of empirical equations considered in the study substantiates the efficacy of the Terzaghi equation in estimating the hydraulic conductivity of river sand-fly ash mix.

Chemotherapeutic Agent-Induced Vulvodynia, an Experimental Model.

Vulvodynia is a chronic clinical condition associated with vulvar pain that can impair the sexual, social, and psychological life of women. There is a need for more research to develop novel strategies and therapies for the treatment of vulvodynia. Vulvodynia in experimental animal models induced via infections, allergens, and diabetes are tedious and with lessor induction rate. The objective of the study was to explore the possibility of inducing vulvodynia using a chemotherapeutic agent in a rodent model. Paclitaxel is commonly used in treating breast and ovarian cancer, whose dose-limiting side effect is peripheral neuropathy. Studies have shown that peripheral neuropathy is one of the etiologies for vulvodynia. Following paclitaxel administration (2 mg/kg i.p.), the intensity of vulvar hypersensitivity was assessed using a series of von Frey filaments (0.008 to 1 g) to ensure the induction of vulvodynia. Vulvodynia was induced from day 2 and was well sustained for 11 days. Furthermore, the induced vulvodynia was validated by investigating the potentiation of a flinch response threshold, upon topical application and systemic administration of gabapentin, a commonly used medication for treating neuropathic pain. The results demonstrate that vulvodynia was induced due to administration of paclitaxel. The fact that chemotherapeutic agent-induced vulvodynia was responsive to topical and parenterally administered gabapentin provides validity to the model. The study establishes a new, relatively simple and reliable animal model for screening drug molecules for vulvar hypersensitivity.

Mitral valve repair in an octogenarian with symptomatic severe mitral regurgitation: First use of MitraClip in India.

Percutaneous mitral valve repair is an accepted treatment of choice in Europe and North America for severe primary or secondary mitral regurgitation, in highly symptomatic patients for whom surgical repair is prohibitively high risk. We describe the first use of the MitraClip in India in a frail elderly female with symptomatic heart failure from severe primary mitral regurgitation who was considered high risk for surgical repair. She had substantial improvement in her symptoms as well as quality of life following the procedure.

Role of Taurine Transporter in the Retinal Uptake of Vigabatrin.

Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1 mM and uptake was significantly increased at pH 7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.

A Rapid Tool to Optimize Process Variables for Continuous Manufacturing of Metronidazole Ointment Using Melt Extrusion Technique.

The use of hot-melt extrusion (HME) technique in the preparation of semi-solid products offers several advantages over conventional processes. However, the optimization of the technique for preparation of semi-solid pharmaceuticals is challenging due to involvement of ingredients with different physical properties. Hence, a simple tool to optimize the mixing of ingredients that results in a target ratio and drug content uniformity is utmost important. In this study, a handheld colorimeter has been explored to optimize the process variables of twin screw processor for preparation of hydrophilic PEG-based ointment. The process parameters which were optimized with use of handheld colorimeter have been used for preparation of polyethylene glycol-based metronidazole ointment. The metronidazole ointment prepared by twin screw processor was compared with commercially available metronidazole gel for in vitro release testing and ex vivo permeation. The flux, ex vivo bioavailability, and Tmax of polyethylene glycol-based metronidazole ointment was found to be similar to that of marketed metronidazole gel.

The identification of differentially expressed genes between extremes of placental efficiency in maternal line gilts on day 95 of gestation.

BACKGROUND: Placental efficiency (PE) describes the relationship between placental and fetal weights (fetal wt/placental wt). Within litters, PE can vary drastically, resulting in similarly sized pigs associated with differently sized placentas, up to a 25% weight difference. However, the mechanisms enabling the smaller placenta to grow a comparable littermate are unknown. To elucidate potential mechanisms, morphological measurements and gene expression profiles in placental and associated endometrial tissues of high PE and low PE feto-placental units were compared. Tissue samples were obtained from eight maternal line gilts during gestational day 95 ovario-hysterectomies. RNA was extracted from tissues of feto-placental units with the highest and lowest PE in each litter and sequenced. RESULTS: Morphological measurements, except placental weight, were not different (P > 0.05) between high and low PE. No DEG were identified in the endometrium and 214 DEG were identified in the placenta (FDR < 0.1), of which 48% were upregulated and 52% were downregulated. Gene ontology (GO) analysis revealed that a large percentage of DEG were involved in catalytic activity, binding, transporter activity, metabolism, biological regulation, and localization. Four GO terms were enriched in the upregulated genes and no terms were enriched in the downregulated genes (FDR < 0.05). Eight statistically significant correlations (P < 0.05) were identified between the morphological measurements and DEG. CONCLUSION: Morphological measures between high and low PE verified comparisons were of similarly sized pigs grown on different sized placentas, and indicated that any negative effects of a reduced placental size on fetal growth were not evident by day 95. The identification of DEG in the placenta, but absence of DEG in the endometrium confirmed that the placenta responds to the fetus. The GO analyses provided evidence that extremes of PE are differentially regulated, affecting components of placental transport capacity like nutrient transport and blood flow. However, alternative GO terms were identified, indicating the complexity of the relationship between placental and fetal weights. These findings support the use of PE as a marker of placental function and provide novel insight into the genetic control of PE, but further research is required to make PE production applicable.

Acute Interstitial Nephritis Following Snake Envenomation: A Single-Center Experience.

OBJECTIVES: To identify the clinical and histopathological characteristics of patients who develop acute interstitial nephritis (AIN) following snake envenomation. METHODS: A retrospective analysis of patients diagnosed with snake envenomation-induced AIN from October 2013 to November 2014. RESULTS: After snake envenomation, 88 patients developed acute kidney injury (AKI). Biopsies were performed on 7 patients due to nonrecovery of kidney function. Among these, 5 patients had AIN. Thus, AIN accounted for 5.7% of snakebite-related acute kidney injury. All patients had severe envenomation at presentation and had prolonged renal failure. Kidney biopsy found a mixed infiltrate composed of predominantly lymphocytes, with variable proportions of other cells including eosinophils neutrophils and plasma cells. The response rate to corticosteroids was 80%. CONCLUSIONS: AIN after snake bite is not uncommon. AIN needs to be considered in patients with persistent renal failure after snake envenomation. Identifying this complication is of utmost importance because of the potentially reversible nature.

Improving polygenic prediction from summary data by learning patterns of effect sharing across multiple phenotypes.

Polygenic prediction of complex trait phenotypes has become important in human genetics, especially in the context of precision medicine. Recently, Morgante et al. introduced mr.mash, a flexible and computationally efficient method that models multiple phenotypes jointly and leverages sharing of effects across such phenotypes to improve prediction accuracy. However, a drawback of mr.mash is that it requires individual-level data, which are often not publicly available. In this work, we introduce mr.mash-rss, an extension of the mr.mash model that requires only summary statistics from Genome-Wide Association Studies (GWAS) and linkage disequilibrium (LD) estimates from a reference panel. By using summary data, we achieve the twin goal of increasing the applicability of the mr.mash model to data sets that are not publicly available and making it scalable to biobank-size data. Through simulations, we show that mr.mash-rss is competitive with, and often outperforms, current state-of-the-art methods for single- and multi-phenotype polygenic prediction in a variety of scenarios that differ in the pattern of effect sharing across phenotypes, the number of phenotypes, the number of causal variants, and the genomic heritability. We also present a real data analysis of 16 blood cell phenotypes in UK Biobank, showing that mr.mash-rss achieves higher prediction accuracy than competing methods for the majority of traits, especially when the data has smaller sample size.

Ear Mites as an Overlooked Source of Ear Itching and Tinnitus - A Case Series.

Otoacariasis is a rare condition characterized by ticks and mites in the ear canal. Human otoacariasis remains underrepresented in literature as otoacariasis is more common in animals. Systemic diseases being transmitted by these arachnids pave the way for potential complications. This case series sheds light on this uncommon condition by highlighting the diverse symptomatology and difficulties in diagnosis and treatment. Three different presentations highlight the diversity of this condition. A 40-year-old male exhibited itching and tinnitus, revealing a mite on the tympanic membrane on otoscopy. A 35-year-old female with persistent itching and tinnitus showed multiple whitish mites on examination. A 50-year-old female complained of ear pain and was found to have a tick attached to the external auditory canal. The relevance and rarity of human otoacariasis are highlighted in this study, thereby encouraging caution in situations of earache. We aim to increase clinician awareness about this condition and the necessary interventions required by conducting a thorough literature review.