Globular PDZ domains typically serve as protein–protein interaction modules that regulate a wide variety of cellular functions via recognition of short linear motifs (SLiMs). Often, PDZ mediated-interactions are essential components of macromolecular complexes, and disruption affects the entire scaffold. Due to their roles as linchpins in trafficking and signaling pathways, PDZ domains are attractive targets: both for controlling viral pathogens, which bind PDZ domains and hijack cellular machinery, as well as for developing therapies to combat human disease. However, successful therapeutic interventions that avoid off-target effects are a challenge, because each PDZ domain interacts with a number of cellular targets, and specific binding preferences can be difficult to decipher. Over twenty-five years of research has produced a wealth of data on the stereochemical preferences of individual PDZ proteins and their binding partners. Currently the field lacks a central repository for this information. Here, we provide this important resource and provide a manually curated, comprehensive list of the 271 human PDZ domains. We use individual domain, as well as recent genomic and proteomic, data in order to gain a holistic view of PDZ domains and interaction networks, arguing this knowledge is critical to optimize targeting selectivity and to benefit human health. Since mutagenesis often affects more than one of these competing factors, it can be difficult to deconvolute the role of pair-wise specificities in controlling the biological read-out of a particular interaction.Stereochemical complementarity is the foundation of molecular recognition. It regulates the formation of protein–protein interactions that govern post-translational modification, trafficking, and localization. In addition to controlling overall molecular activity, the resulting changes in protein chemistry, concentration, and assembly can reciprocally modulate the free-energy landscape of each interaction in its specific physiological context, creating both feed-back and feed-forward systems. Furthermore, the probability of formation of an individual protein–protein interaction This issue is particularly acute in the case of peptide-recognition domains (PRDs), which form the basis for many trafficking and signaling networks within the cell. These domains recognize cognate short linear motifs (SLiMs) – conserved peptide sequence patterns that reflect the stereochemical requirements of corresponding binding pockets in the PRD. Through evolutionary diversification, PRDs typically occur in large families whose distinct binding preferences are driven by sequence variations along the length of the peptide-binding site. However, multiple PRDs may have affinity for a shared target due to incomplete diversification and/or for importance in regulation of biological processes. Conversely, a single PRD often recognizes multiple targets. These domains are often found in tandem with other PRD or functional components, creating a combinatorial mosaic of possible interactions for such multidomain scaffolding proteins. Thus, understanding or manipulating a particular PRD-target interaction often requires detailed biochemical characterization not only of the primary interaction, but also of others within a shared network.PSD-95, a 95kD protein from the post-synaptic density ; Dlg, the Drosophila discs-large-1 tumor suppressor protein; and ZO-1, the epithelial tight-junction protein zonula occludens 1  side chain is bound in an adjacent pocket, whose binding preference is determined not only by the PDZ side chains that line it, but also by the spacing relative to the loop  can underpin physiologically significant interactions , stroke, pain, epilepsy, and depression , cystic fibrosis , neurodegenerative disorders (PICK1), and various cancers  . IdentifA large majority of the available information regarding PDZ domain function has focused on individual domain data. Specifically, work on founding members PSD-95 and Dlg1, as well as Erbin and NHERF1, among others, identified key characteristics, such as known targets, posttranslational modifications, structure, and binding affinities e.g., . While t1.1Estimates of the number of PDZ domains in the human proteome have ranged from 250 to over 400, depending on the database and method of identification used, with a general consensus of approximately 270  and 2α–helices (αA and αB)  . The exaWe therefore superimposed an alignment of the location of the canonical PDZ secondary structure elements onto a sequence alignment of all proposed domains. We also took into consideration whether or not a putative domain contains the conserved carboxylate-binding loop sequence. Of the domains listed by UniProt, 274 contain all required secondary structure elements. However, the carboxylate-binding loop sequence is missing in first PDZ domain of the FERM and PDZ domain-containing protein 2B pseudogene, FRP2L, suggesting that it is not a canonical PDZ domain. In addition, CNIPF is a fusion protein of CNKR3 and IPCEF1, and its PDZ domain sequence is 100% identical with that of CNKR3. Therefore, in analogy to the splice variants mentioned above, we do not consider this to be a unique occurrence of this PDZ domain. Finally, structural alignments identified two candidate PDZ domains that are flagged as distinct in the UniProt list, each representing the second PDZ domain in one of the two Golgi reassembly-stacking proteins GORS1 and GORS2. Although the overall fold is intact, structural data previously revealed a very unusual secondary structure layout, in that the βA and βB strands are at the C-terminal end of the domain, connected to the βE strand . These PTo investigate sequence relationships within this group of proteins, we used CLUSTALW and PHYLIP  to clust0 Ile, as discussed in the next section  of the proteins contain 1 PDZ domain, while 43 (or 28%) contain 2 or more PDZ domains . The numa traces for these 26 available PDZ structures following least-squares superposition of the βB strand and αB helix onto the Rho guanine nucleotide exchange factor 11 (ARHGB) domain. The root-mean-square deviation (RMSD) values are all less than <1.4 Å, independent of binding-motif designation. Areas of tight clustering correspond to the conserved architecture of the domain core around the peptide-binding cleft, in contrast to regions of high structural variability encoded in loop regions. In the surrounding panels, ribbon diagrams for the individual domains are clustered by protein of origin and colored by RMSD of the superposition, permitting visualization of individual adaptations.We selected 20 PDZ domain-containing proteins  and Drosophila melanogaster (fruit fly) PDZ structures, but these sequences have not been included in our table. Searches with a <70% identity cut-off also identify bacterial PDZ domains.Many PDZ domains readily crystallize and hundreds of PDZ domain structures are publicly available. To identify them as comprehensively as possible, we developed a Python-based sequence-matching algorithm utilizing BLASTP . Using 7The set of 471 entries include a total 505 unique structures of PDZ domains. Based on this set, at least one experimental structure has been determined for 163 of the 271 human PDZ domains. The structurally characterized domains belong to 102 of the 154 distinct human proteins containing at least one PDZ domain. Some domains have been extensively characterized: there are 30 distinct PDB entries including structures of DLG4-3, and 29 of the GOPC domain . We noteDrosophila melanogaster Par-6; this mode of binding may be particularly relevant in regulating GPCR signaling , Class II domains recognize X-Φ-X-Φ, and Class III domains recognize X-D/E-X-Φ . In Clas at αB-1 C , and determined binding motifs by testing high affinity interactions using phage display analysis. These extended motifs include PDZ domains Erbin, DLG1-3, INADL-2, and others , Erbin , HtrA1 , HtrA3 , TIP-1 , Tiam1 , and Scrib1 , revealed that ng motif . The thing motif . We founectivity .et al. found that by varying 10 binding site positions on the Erbin PDZ domain using phage display, they were able to generate variants with binding diversity comparable to that of the human PDZ family, including 7 specificity clusters not yet found in nature  system to mutate each residue in the PSD95-PDZ3 domain to each of the other proteogenic amino acids and then to investigate quantitatively how these variants affect ligand binding. They report that 20 out of 81 total sites on the PSD-95 PDZ3 domain functionally affect binding via multiple cooperation networks between residues  Consortium, which provided rigorous benchmarks for computational methods  and isothermal titration calorimetry (ITC) (KD = 787 nM), whereas SPR yielded a much higher-affinity interaction (KD = 10.3 nM), consistent with previously published work  and surface plasmon resonance (SPR) . Importap < 0.05 level. However, the AlphaScreen data are skewed noticeably lower than the other four techniques  reflect avidity effects more strongly than the other techniques, we utilized a linear model, and found significant differences for values determined using AlphaScreen, as compared to the other techniques, including ELISA (p = 0.007), FP (p = 0.006), ITC (p = 0.037), and SPR (p = 0.05).From a mechanistic perspective, the AlphaScreen assay detects the physical proximity of ‘donor’ and ‘acceptor’ beads that are each coated with one binding partner or the other. As a result, both interacting entities are typically multivalent. In comparison, in ELISA and SPR assays, only one binding partner is immobilized on a multivalent surface, while the other is in solution, and in FP and ITC assays, neither partner is immobilized. To test the specific hypothesis that AlphaScreen estimates . We similarly find a statistically significant difference between CAL and PSD-95 PDZ2, both measured by FP , all of which have PDZ binding motif-satisfying E6 proteins . The C-tinocytes . It is i1.70 interacts with residue P0, S−1 with P−1, etc.  . As ment1.8Over the next two sections of this review, we will shift our focus to the regulation of PDZ binding interactions. Outside of the peptide-binding cleft, there are a number of residues that influence target recognition. Indeed, this is due to extensive allosteric networks in PDZ domains. Lockless and Ranganathan first measured energetic couplings that included the Class I αB-1 His residue of PSD-95 PDZ3, residues throughout the core of the protein, and the αA helix, in 1999 . This tyAdditional studies using NMR, kinetic experiments, and extensive mutagenesis on PSD-95 PDZ3, Par-6, PTP-BL PDZ2, and hPTP1E PDZ2 identify residues in the βA-βB and βB-βC loops as modulators of ligand binding , and the tightest interaction was for NHERF1 PDZ1, at 24 nM (An important regulatory mechanism of many PDZ domains is their association with the plasma membrane. Some of the most well studied PDZ domains are those of the membrane-associated guanylate kinase (MAGUK) protein family. Lipid-binding characteristics of PDZ domains have long been studied  by G protein-coupled receptor kinase 5, GRK5  as well as a phospho-SDC1 peptide (pSDC1), with a phosphorylated P−1 Tyr (pY)  . Anotherrylation .2+/calmodulin-dependent protein kinase (CamKII)-dependent phosphorylation of Dlg1 at Ser232, the residue immediately preceding the carboxylate-binding loop sequence, disrupting its interaction with the NMDA receptor subunit GluN2A both in vitro and in Cos7 cells  are comprised almost entirely of a single PDZ domain; however, this is the exception not the norm. A more common feature of PDZ domain-containing proteins is the presence of multiple interaction domains, including additional PDZ domains  Kelil a. The coo+ channel (ENaC) which is crucial for proper Na+/K+ balance. CNKR3 also scaffolds the serine-threonine kinase SGK1 and ENaC inhibitor Nedd4-2, in a >1 MDa complex which plays an important role in Na+ homeostasis  acts as an important regulatory signal by scaffolding the epithelial Naeostasis . In the eostasis . For a reostasis .There are also complexes that consist of multiple PDZ domain-containing proteins. MPP7, for example, forms a tripartite complex with discs large homolog 1 (DLG1) and any of the lin-7 homolog family members (Lin7A-C), drawing together a total of 5 PDZ domains that are important for the proper localization of cell–cell junction components . Due to node as the closest branch point to the central arc of the rooted tree, and distinctly colored the PDZ domain containing-proteins of each node. Our tree agrees with previously reported PDZ families of many members, for example, those of the MAGUK family, characterized by a PDZ-SH3-guanylate kinase “supradomain” architecture , we also need in-depth work on less well studied PDZ domains (e.g., data in Fortunately, there are a number of analogies to aid future work. Kinases, for example, share many SLiM engagement characteristics with PDZ domains. These include the recognition of up to 9 residues in their catalytic site and the use of similar stereochemical and electrostatic mechanisms as PDZ domains . This rePDZ domain target recognition is also very similar to that of SH2 domains, which recognize phosphotyrosine residues downstream of tyrosine kinases. SH2 domain binding motifs are also insufficient to describe these SLiM interactions, and a deeper understanding of modulator-like preferences, both positive and negative, is needed to describe the interactome of each domain . These aAs the basis of future work, we need to start thinking more holistically, treating a combination of signaling domains as one functional entity. There are groups who are doing this, either by cataloguing the interactomes of promiscuous modular domains, then diagramming interaction networks, or by looking at different size scales in the cell that result from macromolecular complexes e.g., . In ordeThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


RL-208 treatment induced an increase in mature brain-derived neurotrophic factor (mBDNF), prevented Tropomyosin-related kinase B full-length (TrkB-FL) cleavage, increased protein levels of Synaptophysin (SYN) and Postsynaptic density protein 95 (PSD95). In whole, these results point out to an improvement in synaptic plasticity. Remarkably, RL-208 also decreased the protein levels of Cyclin-Dependent Kinase 5 (CDK5), as well as p25/p35 ratio, indicating a reduction in kinase activity of CDK5/p25 complex. Consequently, lower levels of hyperphosphorylated Tau (p-Tau) were found. In sum, these results demonstrate the neuroprotectant role of RL-208 through NMDAR blockade.Alzheimer’s disease (AD) is the leading cause of dementia. Non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist memantine improved cognition and molecular alterations after preclinical treatment. Nevertheless, clinical results are discouraging. In vivo efficacy of the RL-208, a new NMDA receptor blocker described recently, with favourable pharmacokinetic properties was evaluated in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD). Oral administration of RL-208 improved cognitive performance assessed by using the three chamber test (TCT), novel object recognition test (NORT), and object location test (OLT). Consistent with behavioural results, RL-208 treated-mice groups significantly changed NMDAR2B phosphorylation state levels but not NMDAR2A. Calpain-1 and Caspase-3 activity was reduced, whereas B-cell lymphoma-2 (BCL-2) levels increased, indicating reduced apoptosis in RL-208 treated SAMP8. Superoxide Dismutase 1 (SOD1) and Glutathione Peroxidase 1 (GPX1), as well as a reduction of hydrogen peroxide (H The prevalence of behavioural abnormalities associated with age-related cognitive decline is growing in older people . Age is The neuropathological hallmarks comprise the accumulation and deposition of β-amyloid (Aβ) in the senile plaques (SNPs). Likewise, the hyperphosphorylation of Tau protein is implicated in neurofibrillary tangles increase (NFTs) . There a2+ to move into the cell for the following physiological functions gates NMDAR. Hence, glutamatergic neurotransmission through NMDAR is critical for neuroplasticity dec-1-yl)methylamine hydrochloride was synthesized as previously described -7-methoxy-4-methylcoumarin (AMMC) for CYP2D6, 7-benzyloxytrifluoromethyl coumarin (7-BFC) and Dibenzylfluorescein (DBF) for CYP3A4) to give the final assay concentrations in a reaction volume of 200 µl. Reactions were terminated after different incubation times, depending on each cytochrome, by the addition of STOP solution (ACN/TrisHCl 0.5M 80:20 and NaOH 2N for CYP3A4 (DBF) and ACN/TrisHCl 0.5M 80:20 for the other cytochromes).Incubations were conducted in 200-µL volume 96 well microtiter plates (COSTAR 3915). Addition of cofactor-buffer mixture (KHFluorescence per well was measured using a fluorescence plate reader (Tecan M1000 pro), and the percentage of inhibition was calculated.SAMP8 is an inbred mouse strain that has been generated by selective inbreeding of the AKR/J strain of mice ,32. It d3,9.04,8]dec-1-yl)-methylamine hydrochloride, (5 mg/kg/day) was administered through drinking water for four weeks before starting the cognitive test. RL-208 was administered up to euthanasia  with 20-weeks-old were used to carry out cognitive and molecular analysis. The animals were randomly divided into four groups: SAMR1 control (SR1 Ct) group (n = 11), SAMP8 control (SP8 Ct) group (n = 8), SAMR1 treated with RL-208 (SR1 RL-208 (5 mg/Kg)) group (n = 10) and SAMP8 treated with RL-208 (SP8 RL-208 (5 mg/Kg)) group (n = 12). Animals had free access to food and water, under standard temperature conditions (22±2ºC) and 12h:12h light-dark cycles (300 lux/0 lux). RL-208,  and by Generalitat de Catalunya .The three-chamber test (TCT) was used to assess preference for social novelty (time spent with a novel intruder in contrast with a familiar one) and sociability (time spent with rodents) . The appThe object location test (OLT) is a well-established task based on the spontaneous tendency of rodents to spend more time exploring a novel object location than a familiar object location, as well as to recognize when an object has been relocated . The tesThe novel object recognition test (NORT) allows evaluating short- and long-term recognition memory involving cortical areas and the hippocampus ,41. The Three days after the behavioural and cognitive tests, mice were euthanized by cervical dislocation. Brains were immediately removed from the skull. The hippocampus and cortex were then isolated and frozen in powdered dry ice. They were maintained at −80 °C for further use. Tissue samples were homogenized in lysis buffer containing phosphatase and protease inhibitors . Total protein levels were obtained, and protein concentration was determined by the method of Bradford. For Western blotting (WB), aliquots of 15 μg of hippocampal protein extraction per sample were used. Protein samples were separated by Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) (8–20%) and transferred onto Polyvinylidene difluoride (PVDF) membranes (Millipore). Afterwards, membranes were blocked in 5% non-fat milk in Tris-buffered saline (TBS) solution containing 0.1% Tween 20 TBS (TBS-T) for 1 hour at room temperature, followed by overnight incubation at a 4 °C with the primary antibodies listed in 2O2 levels from cortex samples were measured as an indicator of oxidative stress, and it was quantified using the Fluorimetric Hydrogen Peroxide Assay Kit (Sigma) according to the manufacturer’s instructions. HTM reagent following the manufacturer’s instructions . The yield, purity, and quality of RNA were determined spectrophotometrically with a NanoDropTM ND-1000  apparatus and an Agilent 2100B Bioanalyzer . RNAs with 260/280 ratios and RIN higher than 1.9 and 7.5, respectively, were selected. Reverse transcription-polymerase chain reaction (RT-PCR) was performed as follows: 2 μg of messenger RNA (mRNA) was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit . Real-time quantitative PCR (qPCR) was used to quantify the mRNA expression of OS and synaptic plasticity genes listed in Total RNA isolation from hippocampal samples was carried out using TRIsureSYBR® Green real-time PCR was performed on a Step One Plus Detection System  employing SYBR® Green PCR Master Mix . Each reaction mixture contained 6.75 μL of complementary DNA (cDNA) (which concentration was 2 μg), 0.75 μL of each primer (which concentration was 100 nM), and 6.75 μL of SYBR® Green PCR Master Mix (2X). Data were analyzed utilizing the comparative cycle threshold (Ct) method (ΔΔCt), where the housekeeping gene level was used to normalize differences in sample loading and preparation . NormaliThe hippocampal determination of pro-Brain-derived neurotrophic factor (proBDNF) and mature brain-derived neurotrophic factor (mBDNF) protein levels was performed using the enzyme-linked immunosorbent assay (ELISA) kit (Biosensis) according to the manufacturer’s instructions. Behavioural analysis was performed blindly, the person who evaluated videos was different from the person who made the behavioural tests. Furthermore, videos are named with a blind code to avoid analysis bias. Data analysis was conducted using GraphPad Prism ver. 7 statistical software. Data are expressed as the mean ± standard error of the mean (SEM) of at least 5 samples per group. Strain and treatment effects were compared using the two-way analysis of variance (ANOVA), followed by Tukey post-hoc analysis or two-tail student’s t-test when it was necessary. Statistical significance was considered when p-values were <0.05. The statistical outliers were determined with Grubbs’ test and when necessary were removed from the analysis. RL-208 was further studied in vitro for ascertaining their microsomal stability and CYP inhibition. RL-208 showed good microsomal stability in rat and mice microsomes, and did not inhibit in a significant way cytochromes CYP2C9, 12 ± 2%, CYP2D6 10 ± 3%; CYP1A2, 5 ± 2%; CYP2C19, 23 ± 1%; CYP3A4 (BFC), 35 ± 4%; CYP3A4(DBF), 3 ± 1%). The TCT assess the general sociability in mice. In the sociability phase, in all experimental groups, the presence of an intruder increased significantly the time spent in the intruder chamber instead of the empty cup chamber A. MoreovNORT evaluation confirmed the cognitive impairment of the SAMP8 mouse model in both short- and long-term recognition memories in comparison with the SAMR1 . StrikinNMDAR changes and apoptotic markers were studied as a learning memory and synaptic plasticity activation. A significant decrease in NMDAR2A protein level was found in SP8 Ct compared to the SR1 Ct A, suggesNext, we evaluated the effects of RL-208 on the proteolytic processes that lead to apoptosis. Calpain-1 and 150 Spectrin Breakdown Products (SBDP) protein levels increased in SP8 Ct in comparison with the SR1 Ct group. RL-208 treatment reduced Calpain-1, Caspase-3 and 120BPD in the SP8 strain, not in SR1 C–E.By contrast, B-cell lymphoma-2 (BCL-2) protein levels diminished, only reaching significance in the SP8 mouse model F. A significant reduction in proBDNF protein levels in SP8 treated mice compared to the control group were found. Differences in SR1 strain were not significant A. ConverTgf) in RL-208 treated mice groups in comparison with the control groups, was observed (Vgf) in SP8 RL-208 compared to the SP8 Ct group, but no changes between SR1 mice groups were found  protein levels were found in RL-208 treated mice A. Postsyobserved C. Likewire found D.Hmox1), an important key enzyme in cellular antioxidant-defense in treated mice (Cox2) in treated mice groups in comparison with the control groups, being significant in the SP8 strain  and Glutathione Peroxidase 1 (GPX1), antioxidant protein levels SP8 mice, but not in SR1 mice A,B. Moreted mice C. Conver8 strain D. Finall8 strain E. ADAM10 protein levels were diminished in SAMP8 in reference to SAMR1, and RL-208 prevented the loss of this secretase A. The CyAs aforementioned, the alteration of NMDAR has been associated with neurodegenerative disorders, such as AD . It has 50, by 1 μM [Early preclinical in vitro experiments indicated that RL-208 displays low IC by 1 μM , has lowSeveral preclinical studies in different transgenic mice models of AD with NMDAR antagonists, including memantine, demonstrate beneficial cognitive activities ,50,51,52The behavioural and cognitive changes induced by RL-208 were accompanied by changes in several molecular pathways associated with NMDAR functionality. RL-208 increased p-NMDAR2B (Tyr1472), which has been proved to play crucial roles in the induction of long-term potentiation (LTP), and the hippocampus-dependent memory formation . There aTgf in the SAMP8 and SAMR1 hippocampus pointed out that the improvement in cognition under pathological conditions was associated with hippocampal plasticity. In the same line, neurotrophic signaling is severely impaired in AD, being BDNF/TrkB representative signaling pathway altered. BDNF/TrKB signaling pathway was ameliorated under RL-208 treatment, with significant increases in mBDNF, TrKB-FL, and SNAP25 protein levels. These results could indicate that RL-208 contributes to restoring cognition in SAMP8 mouse because of the increase in BDNF/TrKB signaling. Previous studies have been described as the ability of NMDAR antagonists to modify synaptic plasticity ,56,57. IHmox1 gene expression increased, as well as Cox-2 gene expression diminished in SAMP8 after RL-208 treatment. Then, in line with our findings, RL-208 delivered a significant diminution in OS because of its role as uncompetitive NMDAR antagonist.It has been reported that OS is involved in several neurodegenerative disorders, mediating neuronal death . SeveralTau hyperphosphorylation is a characteristic histological mark in several neurodegenerative disorders. Tau phosphorylation levels are regulated by a complex network of protein kinases and phosphatases, such as CDK5 . RL-208 In sum, our in vivo study in SAMP8 demonstrated the therapeutic potential of RL-208, a novel NMDAR uncompetitive antagonist, for age-related cognitive decline, and AD. Because it is of interest to shed light on the underlying mechanisms by which NMDAR mediates neuroprotection and new pharmacological approaches are needed to fight devastating neurodegenerative diseases as AD, RL-208 beneficial effects described here, offer new clues to face the challenge in drug discovery . Notewor


In a real-world application, the images taken by different cameras with different conditions often incur illumination variation, low-resolution, different poses, blur, etc., which leads to a large distribution difference or gap between training (source) and test (target) images. This distribution gap is challenging for many primitive machine learning classification and clustering algorithms such as k-Nearest Neighbor (k-NN) and k-means. In order to minimize this distribution gap, we propose a novel Subspace based Transfer Joint Matching with Laplacian Regularization (STJML) method for visual domain adaptation by jointly matching the features and re-weighting the instances across different domains. Specifically, the proposed STJML-based method includes four key components: (1) considering subspaces of both domains; (2) instance re-weighting; (3) it simultaneously reduces the domain shift in both marginal distribution and conditional distribution between the source domain and the target domain; (4) preserving the original similarity of data points by using Laplacian regularization. Experiments on three popular real-world domain adaptation problem datasets demonstrate a significant performance improvement of our proposed method over published state-of-the-art primitive and domain adaptation methods. Indoor–outdoor camera surveillance systems ,2 are wiRecently, the literature ,4 has seIf the distribution difference between both domains is large enough, there will always be a situation where the source domain instances are not relevant to the target domain instance, even after finding a common feature location. In this situation, jointly optimizing instance re-weighting and feature matching is an important and unavoidable task for robust transfer learning. To understand a need for joint-learning instance re-weighting and feature matching more deeply, let us consider an example in which we have source domain data with outlier data samples (or irrelevant instances) as shown in Fortunately, in the literature, there is a method called Transfer Joint Matching (TJM) that performs joint feature matching and instance re-weighting by down-weighting irrelevant features of the source domain . HoweverIn this paper, to solve all of the above-discussed challenges and to efficiently transfer knowledge from the source domain to the target domain, we propose a novel Subspace based Transfer Joint Matching with Laplacian Regularization (STJML) method for visual domain adaptation by jointly matching the features and re-weighting instances across both the source and the target domains.The proposed method STJML is the first framework that crosses the limits of all the comparative cutting edge methods, by considering all inevitable properties such as projecting both domain data into a low dimensional manifold, instance re-weighting, minimizing marginal and conditional distributions, and geometrical structure of both domains in a common framework.With the help of the t-SNE tool, to illustrate the reason for the inclusion of all the components (or inevitable properties), we have graphically visualized the features learned by the proposed method after excluding any component.The major contributions of this work can be listed as follows:Recently, various DA and TL approaches have been proposed for transferring structure or information from one domain to another domain in terms of features, instances, relational information, and parameters ,9. HowevIn the first type, the objective is to minimize the distribution difference between the source and the target domains based on feature learning. For example, Pan et al.  proposedIn the second type, the objective is to re-weight the domain samples so as to minimize the distribution difference between both domain samples. The TrAdaBoost TL  method rIn the final type, the target domain metric is to be learned by establishing a relationship between the source domain and the target domain tasks. Kulis et al.  introducThis section presents the Subspace based Transfer Joint Matching with Laplacian Regularization (STJML) method in detail.x. This classifier function To understand transfer learning or domain adaptation, the domain and the task must be explicitly defined. A domain Transfer learning, given a labeled source domain Z for both domains, perform feature matching, instance re-weighting, and the similar geometrical property exploitation in a Reproducing Kernel Hilbert Space (RKHS) to match both first and high-order statistics.To address the limitations of existing TL methods, the STJML method minimizes the distribution gap statistically and geometrically by working on the following components: finding both domain subspaces, matching features, instance re-weighting, and exploiting the similar geometrical property. In our proposed STJML approach, first, we exploit the subspaces of both domains and then with the help of common projection vector-matrix D-dimensional feature space, they are drawn according to different marginal distributions. Consequently, according to However, even though maximizing the subspace data variance, the distribution difference between both domains will still be quite large. Therefore, the main problem is to minimize the distribution difference between them by applying an appropriate distance metric (or measure). There are many distance measures (such as the Kullback–Leibler (KL) divergence) that can be utilized to compute the appropriate distance between both domain samples. However, many of these methods are parameterized or require estimating the intermediate probability density . TherefoMinimizing the marginal distribution difference does not guarantee that the conditional distribution between both source and target domains will also be minimized. However, for robust transfer learning, minimizing the conditional distributions, i.e., required . Reducincth class in the source domain, cth class in the target domain, Long et al.  proposedV will be more robust till its convergence. As there is a difference in both the marginal and conditional distributions, the initial By minimizing Equation  such thaHowever, matching features with marginal and conditional distributions is not sufficient for transfer learning, as it can only match first- and higher-order statistics. In particular, when the domain difference is significant enough, even in the feature learning, there will always be some source instances or samples that are not related to the target instance. In this condition, an instance re-weighting method with feature learning should also be included to deal with such a problem.Z. Because each entry of the matrix Z corresponds to an example, row sparsity can substantially facilitate instance re-weighting. Thus, instance re-weighting regularizer can be defined as follows.In this paper, we adopt a posed in . This refollows.Ir=Zs2,1+However, matching features and instance re-weighting are not enough to convey knowledge transfer by capturing the intrinsic structure of the source domain labeled samples and target domain unlabeled samples. In particular, labeled data samples of the source domain combined with unlabeled data samples of the target domain are used to construct a graph that sets the information of the neighborhood data samples. Here, the graph provides discrete approximations to the local geometry of the manifold data. With the help of the Laplacian regularization term s labels .K, we can use a Given a kernelized data matrix L is the Laplacian matrix; Here, two data samples are connected with an edge if they are likely to be from the same class. Thus, the regularizer term The objective of this work is to minimize the distribution difference between domains by jointly matching the features of both domains and re-weighting the source domain samples, and preserving original similarity of both domain samples. So, by incorporating Equations , 5), , , the proibutions , η is thZ, we partial derivative Z and equate it to zero asBy using the Lagrange multiplier Equation  can be wAs the problem in Equation  is a genIn this section, we present a performance of the proposed STJML method by experimenting on various visual domain classification problems.We have considered three publicly image datasets: Office + Caltech10 with Speeded Up Robust Features (SURF), Office + Caltech10 with VGG-FC6 features, and Pose, Illumination, and Expression (PIE) face Recognition for experimentation. These data sets are well known in the domain adaptation methods and are widely considered in most recent works such as ,26)..26]).Caltech-256 has 30,607 images and 256 classes, while Office-31 is made of three object domains: DSLR (D), Amazon (A), and Webcam (W). It contains a total of 4652 images with 31 classes. As images in Office and Caltech-256 having different distributions, DA methods can help with cross-domain recognition. Since both the datasets contains 10 common classes, we considered Office + Caltech 10 datasets from , which hCarnegie Mellon University (CMU) PIE ) face dataset  containsP to the target domain Q.In this paper, we use notation In order to visualize the learned feature space by our proposed method, we considered the t-SNE tool , throughTo quantify misclassification samples in the learned feature space by the proposed method STJML for the task  : TCA is Joint Distribution Alignment (JDA) : This meGeodesic Flow Kernel (GFK) : In ordeTransfer Joint Matching (TJM) : This meSubspace Alignment (SA) : SA firsScatter Component Analysis (SCA) : This meCORAL : It aligAdaptation Regularization (ARTL) : This meCross-Domain Metric Learning (CDML) : It is aClose yet Discriminative DA(CDDA), Geometry Aware DA (GA-DA), and Discriminative and Geometry Aware DA (DGA-DA) : CDDA enInvariant Latent Space (ILS) : This TLBalanced Distribution Adaptation (BDA) : It is aJoint Geometrical and Statistical Alignment (JGSA) : It exteRobust Transfer Metric Learning (RTML) : This meDomain Invariant and Class Discriminative (DICD) : This DAExplicit Map-based Feature Selection (EMFS) : It atteDomain Irrelevant Class clustering (DICE) : This meLinear Discriminant Analysis-inspired Domain Adaptation (LDADA) : The keyKernelized Unified Framework for Domain Adaptation (KUFDA) : This TLThe proposed STJML method was verified and compared with many state-of-the-art primitive and domain adaptation algorithms. A brief description of all the comparative methods is as follows:k from 1 to 10 with an interval of 1 and keep other parameter values Our proposed STJML method contains various parameters such as  methods ,8,13,16. methods ,40, we ak for each task. Therefore, we need to find out its proper value for each task of the datasets. For each task of the considered datasets, we varied k value from 1 to 10 with an interval of 1 and keep other parameter values constant as shown in In our proposed method, we considered the k-NN classifier to predict the label of the target domain, and the performance of this classifier depends on an appropriate value of parameter k, we require an appropriate value of parameter Similar to the parameter This parameter quantitatively evaluates the importance of aligning marginal and conditional distributions in domain adaptation. In this evaluation, the existing work ,8 in DA As parameter Similar to the parameter As the performance of the proposed STJML method depends on choosing the eigenvectors  and testing (target data) datasets.Among domain adaptation methods, the GFK method’s performance is worse for an average accuracy of all tasks in the Office + Caltech dataset for both SURF and VGG-FC6 features.The JDA method’s performance for all the three datasets is higher than that of the TCA method because it adopts the conditional distribution in addition to the marginal distribution.The ILS method works well compared to other subalignment methods  because of considering the more robust discriminative loss function for the Office + Caltech dataset with deeper features.As TJM adopts the term instance re-weighting, its performance is better than other DA methods such as TCA, GFK, JDA, SA, CORAL, ILS, and BDA for the deep features of Office + Caltech dataset. However, for the SURF features, TJM gives better average accuracy than SCA, ARTL, GFK, and TCA, but performs poorly compared to JGSA, CORAL, LDADA, DICE, RTML, ILS, and JDA.The average accuracy (65.09%) of the DGA-DA method for all tasks in the PIE face dataset is higher than that of other methods  because it involves novel repulsive force term and the Laplacian regularization term.Since JGSA improves JDA by considering two projection vector matrices and preserving source domain discriminant information, the mean accuracy (82.60%) of the JGSA method for all tasks in the Office + Caltech dataset with deep features is higher than other methods . Similarly, for the Office + Caltech dataset with SURF features, it performs well compared to all other comparative methods except KUFDA and DICE.As KUFDA improves JGSA by considering the term Laplacian regularization, its average accuracy is much higher than other methods for the deep features of the Office + Caltech dataset, but less than the DICE method for most of the tasks in the PIE face dataset.For the PIE face and the Office + Caltech10 with SURF features datasets, DICE is performing better than all other methods (except STJML) because it is taking care of the intra-domain structure, especially for the target domain. However, its performance is abysmal for deep features of the Office + Caltech 10 dataset.Since our proposed method covers all the important objectives, as well as works on the projected subspaces of both the domains, the average accuracy of the proposed STJML method for all the tasks in all the considered datasets, is higher than all the other comparative methods. However, KUFDA beats our proposed algorithm for some tasks in the Office + Caltech dataset with deep features such as The recognition performance of the proposed method and the other compared state-of-the-art methods on three widely used domain adaptation datasets is reported in Algorithm 1: Subspace based Transfer Joint Matching with Laplacian Regularization (STJML)Here, we analyze the computational complexity of Algorithm 1 by the tth iteration. Therefore, its computational cost is higher than the TCA and GFK methods. The TJM method enhances JDA performance by considering additional instance-weighting terms. Thus, the computation cost of TJM also increases compared to JDA. Since the CORAL method first finds the co-variance matrices for both domains and then aligns the source domain to the target domain, its computation cost is higher than that of TJM, JDA, GFK, and TCA. JGSA includes two additional terms such as discriminative terms and subalignments and also uses two projection vector matrices for both domains. Therefore, JGSA consumes much time compared to other TJM, JDA, GFK, CORAL, and TCA. Since our proposed method improves the TJM by considering the subspace of both domains as well as adding the Laplacian term, it has a higher cost than TJM, but lower than JGSA.To analyze the actual running time taken by each algorithm, we considered three tasks for the experiment such as In this paper, we proposed a novel Subspace based Transfer Joint Matching with Laplacian Regularization (STJML) method for efficiently transferring knowledge from the source domain to the target domain. Because of jointly optimizing all the inevitable components, the proposed STJML method is robust for reducing the distribution differences between both domains. Extensive experiments on several cross-domain image datasets suggest that the STJML method performs much better than state-of-the-art primitive and transfer learning methods.In the future, there are several ways through which we can extend our proposed method STJML, and some of them are:Firstly, we will extend the STJML method to multi-task learning environments , where mSecondly, since the STJML method has many parameters and conducting manual parameter sensitive tests to find appropriate values is a hectic and time-consuming process. Furthermore, the STJML method uses the original features to find a common feature space. Still, the original features itself are distorted, then the STJML method will not become a robust classifier. Therefore, in the future, we will use the particle swarm optimization  method tLastly, nowadays, there is increasing interest in neural-network-based learning models  due to t


Optimisation using the response surface methodology showed ash (0.79–4.42%) and crude fibre (2.77–5.12%) increased as the drying temperature (DT) and concentration of pretreatment (COP) increased. Starch content (78.06–84.71%) was not influenced by the processing variables. Both pretreatments improved the lightness and whiteness index of cassava flour. Optimal processing conditions of 70°C DT and 3%w/v COP were the same for the proximate composition of cassava flour from all experimental groups.Calcium chloride and citric acid (0.6–3.4% Manihot esculenta Crantz), which is also known as tapioca or manioc [Euphorbiaceae. The tuberous roots, rich in starch, are the main storage organ in cassava and the major part of the plant that is mostly consumed [The world's most important staple root crop is cassava  , 7. Rapiw/v) are separately applied in the pretreatment of flour from two South African cassava landraces (white and red) under drying temperatures of 45–74°C. RSM is employed to determine the linear, interactive, and quadratic effects of these processing variables on the colour properties  and proximate components  of cassava flour.Processing of cassava flour involves several units which can be varied as the quality of flour is determined by the process it undergoes . ResponsTwo landraces of cassava (white and red) were sourced from the Institute of Tropical and Subtropical Crops—Agricultural Research Council (ITSC-ARC), Levubu, Limpopo Province, South Africa . The roots were harvested fourteen months after planting and sorted before washing with tap water generously to remove adhering dirt and avoid contamination during processing. Within 24 h after harvest, processing commenced after ascertaining that the roots were in a fresh state, before the onset of deterioration .L∗, a∗, b∗, chroma, colour difference, whiteness index, and brownness index) of cassava flour. Each experimental run generated by the DE software was done in triplicate.Before the conduct of the experiment, Design Expert (DE) software version 11 generated the experimental conditions for processing the flour samples. The conduct of the experiment was done using the central composite design with two independent variables: concentration of pretreatment (COP) and drying temperature (DT). μm sized aperture sieve and stored, till further analysis, in sample bags made of paper material at room temperature. Two control samples were processed, one from each landrace, using the same procedure as the experimental samples save for pretreatment and DT was 60°C. The control samples served as reference samples to experimental samples during statistical analysis within a landrace.The method described by the Federal Institute of Industrial Research  was emplL∗ (lightness/darkness), a∗ (redness/greenness), and b∗ (yellowness/blueness). The colorimeter was calibrated with a standard white  and black plate before use. Chroma, colour difference (ΔE), whiteness index (WI), and brownness index (BI) were calculated as follows [x = (a∗ + 1.75L∗)/(5.645L∗ + a∗ − 3.012b∗).A colorimeter  was employed in the determination of the following colour properties of the samples:  follows :(1)ChroPercentage ash, crude fibre, moisture, and starch contents of the samples were measured using an NIR analyser . The analyser's open-faced sample dish was filled with cassava flour and placed in the NIR analyser. The samples were scanned by infrared rays emitted by the machine, and the software on the PC displayed the readings obtained. The NIR analyser was previously standardised by a selection of calibration and validation samples and reference data obtained by routine laboratory analysis .R2), p values, F values, response surface plots, contour plots, and optimisation conditions were generated by the software for all four experimental groups. Statistical data analysis was done with the IBM SPSS Statistics software version 25  to ascertain the significant difference between means of the experimental and reference/control samples within each experimental group at a 95% confidence level. One-way ANOVA and separation of means were done using Duncan's multiple range test.Design Expert software version 11 was employed in the analysis of variance (ANOVA) of the linear, interactive, and quadratic effects of the model parameters on cassava flour properties. Regression models, coefficient of determination  of the ash content were relatively high between 0.9446 and 0.9710, which show that the model fits.The ash content of cassava is an indication of its nonvolatile content and mineral richness . All fouX2 (COP) axis. A slight decrease in moisture was observed with an increase along the X1 (DT) axis. ANOVA of model parameters produced p values less than 0.05 for the linear effect of DT and quadratic effect of COP. Regression models yielded a coefficient of variation of 0.8631–0.8990, and all had positive intercepts. Materials, like flour, having above 12.5% moisture possess less storage stability than those with less moisture content; hence, a moisture content of not more than 12.5% is generally specified for flours [High moisture content in foods aids the growth of microbes ; hence, r flours . The amow/v) and 4 (70°C/3%w/v) in all the experimental groups. It gives a clue that the crude fibre content increased with DT and COP. The response surface plots for all experimental groups (p < 0.05230) the fibre content of the flours. The high fibre content obtained in this study may positively influence the dietary fibre content available in the flours. Consumption of an ample quantity of dietary fibre decreases the risk of diseases such as obesity, constipation, gallstones, diabetes, and coronary heart diseases [Textural behaviour and in vitro digestibility of cassava flour are influenced by its residual fibre . The crul groups  align wid 4 70°C/%w/v in al groups  as is dodiseases . This isp < 0.05) across all experimental groups. The F values (<0.01–15.15) in p values below 0.05 for the model and linear effect of COP for samples pretreated with citric acid from the white landrace (CAW). This is evident in the response surface plots  L∗, a∗, b∗, chroma, whiteness index (WI), brownness index (BI), and change in colour (ΔE). Regression models relating the colour properties to actual levels of processing conditions are depicted in E—CAR) and 0.9981 (b∗—CCW).Colour is a physical yet vital parameter that determines consumers' preference for a product. Tables L∗ values of the cassava flour samples were between 91.37 and 93.65 with the control sample significantly lower (p < 0.05) than the experimental samples in all four groups. This shows that pretreatment of cassava flour from both landraces with citric acid or calcium chloride enhances the lightness of the flour. Soaking and grating were reported to cause a significant (p < 0.05) increase in the L∗ values of cassava flour [L∗ values (not reported) in all experimental groups and the increase of L∗ values along the COP axis of the response contour plots (not reported) buttresses the fact that pretreatment improved the lightness of the flour. These findings are in line with the report of Nemaungani et al. [va flour . The sigi et al.  on citria∗ value) and blueness/yellowness  of the flour samples ranged from -0.52 to 0.97 and from 4.55 to 8.24, respectively. Hasmadi et al. [a∗ values (-0.11 and 0.63) and b∗ values (4.55 and 8.24) for flour processed from two cassava varieties grown in Sabah, Malaysia. The ranges of a∗ (-1.75 to -0.74) and b∗ (6.42 to 9.92) values reported by Chiwona-Karltun et al. [a∗ values of the control sample in citric acid pretreated flours were significantly lower (p < 0.05) than the experimental samples within the respective groups  is a function of the difference between the L∗, a∗, and b∗ values of the experimental samples and the control. Colour change was the highest (9.54–9.69) in cassava flour processed from the white landrace pretreated with citric acid (CAW) and the lowest (1.11–1.81) in cassava flour processed from the red landrace pretreated with citric acid (CAR). This gives a hint on the effect of landrace on the colour properties of cassava flour. As for the whiteness index (WI), the trend was similar to that of the L∗ value. The control in all experimental groups had the least WI. The values of L∗ (87.67–93.57), a∗ (-0.27–1.1), b∗ (8.4–11.83), chroma (8.4–11.87), and WI (82.88–89.42) reported in the work of Omolola et al. [L∗, a∗, and WI increased while b∗ values decreased with citric acid modification of yam starches from four different cultivars.Change in colour  for all four experimental samples were the same. However, the responses and desirability differed for CAR , CCR , CAW , and CCW .Multiresponse optimisation of proximate components and colour properties of cassava flour was conducted, separately, using the Design Expert software. The following optimisation goal for the former was set: starch content in range, minimised moisture content, and maximised ash and crude fibre contents. Overlay plots for optimal processing conditions for cassava flour from all four experimental groups with the desired qualities are shown in L∗ values. The two maximised colour parameters correspond to consumers' preference for cassava flour. Omolola et al. [w/v and 0.795 desirability; CCR—57.31°C/2.45%w/v and 0.546 desirability; CAW—50°C/3%w/v with desirability of 0.846; and CCW—51.87°C/2.95%w/v and 0.821 desirability. Low a∗ and b∗ values with relatively high L∗ and WI values recorded for all flour samples indicate that the samples are white. Whiteness is an advantageous consumer property especially for substitution of wheat flour.Optimisation of colour properties had the following targets: minimised BI and maximised WI and a et al.  stated tw/v) and drying temperature (45–74°C) in cassava flour processing. The study showed that the processing conditions yielded cassava flours with good shelf stability, increased ash content, and high fibre content which is good for the health of consumers. An increase in lightness and whiteness index of the flour indicates that both calcium chloride and citric acid pretreatment has mitigating efficacy against enzymatic browning associated with PPD and processing of cassava flour. The chemical pretreatment and drying temperatures had no significant effect (p > 0.05) on the starch content of cassava flours. Response surface plots, multiresponse overlay optimisation plots, and regression models generated from this study assist in eliminating the preexisting gap of the interactive effect of pretreatment and drying temperature on proximate composition and colour properties of cassava flour.Application of response surface methodology was effective in investigating the linear, quadratic, and interactive effects of calcium chloride or citric acid pretreatment (0.6–3.4%


Persistent left superior vena cava (PLSVC) is the most common thoracic venous anomaly and may be a component of the complex cardiac pathologies. While it is often asymptomatic, it can lead to significant problems such as arrhythmias and cyanosis. Besides, it can cause serious complications during vascular interventional procedures or the surgical treatment of cardiac anomalies (CA). The clinical significance of PLSVC depends on the drainage site and the accompanying CA. In this article, we will describe the epidemiology, embryology, and anatomic variations of PLSVC. Possible accompanying CA and heterotaxy spectrum will be reviewed with the help of multidetector computed tomography (MDCT) images. Radiological pitfalls, differential diagnoses, and the clinical importance of PLSVC will be highlighted. Persistent left superior vena cava (PLSVC) may lead to significant clinical symptoms and may affect surgical management.PLSVC may accompany various congenital cardiac diseases as well as heterotaxy spectrum.To be aware of the differential diagnoses of PLSVC is essential for correctly interpreting left-sided mediastinal vascular structures.Persistent left superior vena cava (PLSVC) is a rare vascular anomaly that begins at the junction of the left subclavian and internal jugular veins, passes through the left side of the mediastinum adjacent to the arcus aorta. It mostly drains into the right atrium via the coronary sinus (CS). Although PLSVC is infrequent among all vascular anomalies, it is the most common thoracic venous anomaly. Mostly, PLSVC is asymptomatic and detected incidentally in diagnostic and therapeutic examinations due to different reasons. However, it can be discovered as a component of the complex cardiac pathologies and may lead to significant problems such as arrhythmia –4.There are different modalities for evaluation of PLSVC, such as perinatal echocardiography, multidetector computed tomography (MDCT), magnetic resonance imaging (MRI), and invasive angiography. The advantages, disadvantages of these modalities, and optimal techniques for imaging of PLSVC are shown in Table In this article, we will describe the epidemiology, embryology, and anatomic variations of PLSVC. Possible accompanying cardiac anomalies (CA) and heterotaxy spectrum will be reviewed with the help of MDCT images. The radiological pitfalls with their CT imaging features that may help make the differential diagnosis, and the clinical importance of PLSVC will be highlighted.The exact frequency of PLSVC is not known because PLSVC is often asymptomatic and is detected incidentally. There is no significant difference in its prevalence between males and females. The prevalence of PLSVC ranges from 0.2 to 3% in the general healthy population. In patients with congenital heart disease (CHD), its prevalence ranges between 1.3 and 11%. Additionally, the prevalence of PLSVC is thought to be higher in the prenatal period since the accompanying anatomic anomalies, including heart defects, may cause spontaneous abortions and premature deaths , 9.The primitive venous system consists of three paired veins: vitelline veins (VV), umbilical veins (UV), cardinal veins (CV). Superior and inferior CVs are essential structures that allow the blood to return from the cranial and caudal parts of the embryo to the primitive heart. They combine to form common CVs (or the duct of Cuvier) draining into the double horned sinus venosus , 3, 9. TDifferent hypotheses regarding the development of PLSVC have been proposed. One of these is “low left atrial pressure theory.” According to this theory, in the presence of anomalies, which may cause reduced left atrial pressure and insufficient development of the left atrium, such as atrioventricular septal defect (AVSD), the left atrium will be smaller than expected. Thus, it will not be able to compress the CS and left CVs adequately. As such, the left common CV and caudal part of the left superior CV will not regress, and PLSVC will develop. Some hypotheses suggest the vice versa. According to the “obstructive theory” hypothesis, the presence of PLSVC, which may cause an increase in CS size, could lead to the formation of a left-sided obstructive lesion because of the space restriction .PLSVC is responsible for approximately 20% of the total venous blood return from the left arm, left half of the head and neck. The right atrial drainage is seen in 80–90% of cases, while the left atrial drainage accounts for the remaining 10–20%. Generally, it joins into the right atrium through the CS and mostly has no hemodynamic effect. However, CS ostial atresia may accompany PLSVC. In that case, PLSVC becomes the major retrograde drainage pathway for coronary veins unless collateral drainage pathways develop between the coronary sinus and the heart chambers. The left atrial drainage, which is rare, occurs directly via the left atrial appendage or indirectly through the left pulmonary veins or the CS. In some sources, the latter is defined as an unroofed CS or CS atrial septal defect. The association of the atrial septal defect (ASD) and PLSVC draining into the left atrium via unroofed CS is called as Raghib syndrome  accompanies PLSVC, and this situation is known as double SVC (DSVC). If the caudal part of the right superior CV regresses in the intrauterine period, RSVC cannot develop, resulting in the presence of isolated PLSVC (IPLSVC). Mostly, IPLSVC is associated with CA and cardiac situs disorders. However, there are examples of IPLSVC without any accompanying apparent CA in the literature. In cases of DSVC, dimensions of RSVC may be larger or smaller than PLSVC Fig. a–c 2, 33, 9, 13.In 65% of the cases, DSVC runs along each side of the mediastinum without interconnection. However, there could be the left brachiocephalic vein (LBCV) connecting them, which is also called the bridging vein (BV) Fig. d, e 3]..3].To date, many CA associated with PLSVC have been identified and grouped in different ways , 14, 15.In the literature, there is a wide range of information about the frequency of cardiac anomalies accompanying PLSVC , 14–22. Moreover, attention has been drawn to the relationship of some specific cardiac anomalies with PLSVC in many publications. In addition to left-sided pathologies such as mitral atresia, cor triatriatum, and hypoplastic left heart, transposition of the great arteries (TGA) and tricuspid atresia are other rarer anomalies that have been reported to be closely related to PLSVC in the literature , 16, 20.Different cardiac anomalies come to the fore in different situations such as type of accompanying cardiac anomaly (cyanotic or acyanotic), presence of heterotaxy, and drainage location of PLSVC , 15, 21.Association of PLSVC with aorta-related pathologies such as right-sided arcus aorta (RAA) and coarctation of the aorta (CoA) have also been emphasized in the literature. It was mentioned that the association of PLSVC with RAA is approximately 16% . In anotThe term heterotaxy comprises situs inversus and situs ambiguus (right/left isomerism) Fig. . DSVC orAccording to the literature, PLSVC-heterotaxy coexistence is frequently observed, and PLSVC is present in 50–70% of heterotaxy cases. Additionally, it is informed that 45% of patients with PLSVC in the antenatal period have accompanying heterotaxy . In a stAccording to a study, while right atrial isomerism in patients with PLSVC is about 7%, left atrial isomerism is about 9% . In anotComplete atrioventricular septal defect, right ventricular outflow tract obstruction (RVOTO) (pulmonary stenosis and atresia), and double outlet right ventricle (DORV) are found as the most common accompanying anomalies of PLSVC in patients with heterotaxy  Table 4. The preBerg et al. reported that they never saw CS dilatation, which is a well-known sonographic finding supporting the presence of PLSVC, in the heterotaxy group. The absence of CS dilatation has been associated with unroofed CS, which is found in almost all heterotaxy cases. It should be kept in mind that the absence of CS dilatation does not exclude the presence of PLSVC in patients with heterotaxy during the antenatal period, and the possibility of concomitant unroofed CS anomaly is high .The clinical significance of PLSVC depends on the drainage site and the accompanying anomalies. PLSVC without CA is generally asymptomatic and is detected as an incidental finding. In the case of PLSVC with right atrial drainage, the CS often expands Fig. g, h. ThiIn a recent study by Yun Gi Kim et al. , it was It is crucial to know the PLSVC existence in advance in invasive procedures, such as central venous catheter (CVC) insertion Fig. a, b, carThe presence of CS ostial atresia is also critical in the operations that require PLSVC ligation. In this case, the CS still drains the blood from the coronary veins to the right atrium via the retrograde PLSVC-LBCV-RSVC pathway, instead of the atretic ostium. The ligation of PLSVC will be catastrophic due to the acute interruption of the cardiac venous drainage .The left atrial drainage of PLSVC Fig. d–f, someThe knowledge of PLSVC is fundamental in some cardiac surgeries such as venous rerouting procedures, operations with cavo-pulmonary anastomosis , and heart transplantation. In heart transplantation surgery, if PLSVC without BV is present in the recipient’s heart, the bicaval anastomosis technique will be performed. It requires separation of the CS of the donor’s heart for the establishment of the recipient’s PLSVC anastomosis to the donor’s right atrium .In the case of unknown PLSVC, retrograde cardioplegia, a common practice for cardiac surgeries for myocardial protection, will be ineffective. Clamping of PLSVC may be required for the prevention of retrograde flow. However, cardioplegia may fail even after clamping of PLSVC, due to the steal effect by the hemiazygos venous system linked to PLSVC , 3.During cardiopulmonary bypass, not knowing PLSVC existence may result in both surplus blood return through the right atrium and insufficient venous return to the pump. This problem is mostly encountered in pathologies such as pulmonary atresia, tricuspid atresia, TOF, where increased systemic venous pressure gets over the level of left atrial pressure .With the help of screening echocardiography, PLSVC can be detected as early as in the prenatal period. It can be used as a marker for cardiac or non-cardiac embryopathy. It may require extensive evaluation to exclude possible developmental anomalies. In cases with CHD, symptoms will be mainly due to these anomalies .In the presence of the vessel on the left side of the aorta in the mediastinum, other vascular structures apart from PLSVC should be considered in the differential diagnosis. They are vertical vein, levoatriocardinal vein, left superior intercostal vein, aberrant left brachiocephalic vein, pericardiophrenic vein, and vascular structures secondary to surgery.To make the definitive diagnosis, features which should be taken into consideration are as follows: “origin site,” “drainage site,” “orientation of the route between the origin and drainage site according to mediastinal structures,” “the expected direction of the blood flow,” and “characteristics of accompanying cardiac and non-cardiac diseases.” According to the above-mentioned features, a comprehensive summary of the differential diagnoses of PLSVC is depicted in Fig. Some masses on the expected course of PLSVC could be confusing at first look due to their location. For making the differential diagnosis, it is essential to follow all of the slices carefully and see the beginning and end of the mass Fig.  25, 26], 2625, 2Moreover, an interesting variant of PLSVC, which has an intra-atrial course within the left atrium, has been identified recently. If this pitfall variant is not known, it may be misunderstood as left atrial cystic mass, may cause patient anxiety, and may lead to unnecessary effort for further investigations .The vertical vein (VV) is the vessel that drains the blood from the pulmonary veins into the LBCV in the presence of supracardiac type total or partial APVD (TAPVD or PAPVD) Fig. . It may There are also some auxiliary features to differentiate PLSVC and VV. The expected flow direction is craniocaudal in PLSVC, while it is caudocranial in the VV. In the case of PLSVC, there are two vessels in the anterior aspect of the left main bronchus: one of them is PLSVC, and the other one is the left superior pulmonary vein. Ordinarily, only the left superior pulmonary vein is expected to be at this location. However, in the case of the VV with PAPVD, no vessel is seen in the anterior aspect of the left main bronchus. The size of the LBCV can also be helpful in finding for differentiation. In the case of APVD, the LBCV and RSVC may be of large caliber because the VV transports blood via these venous structures. On the other hand, PLSVC, frequently, is associated with an absent or small-sized LBCV , 28.The levoatriocardinal vein (LACV) is the interatrial connection that originates from the left atrium (68%) or pulmonary vein (32%). It drains into one of the systemic venous structures, mostly, into the LBCV (48%) Figs.  and 19 , 30.FigThe differentiation of LACV from PLSVC with right atrial drainage is straightforward. Because this drainage site is unlikely for the levoatriocardinal vein. Similarly, in cases where PLSVC drains into the left atrium via the unroofed CS, unroofed CS and ASD facilitate the differential diagnosis in favor of PLSVC, since they are unusual for LACV –31.However, PLSVC may drain directly into the left atrium or pulmonary vein. In this situation, the expected origin and drainage site of those two vessels will be the same, and it is necessary to search other features for distinguishment. The anatomical feature that may help distinguish is their relative orientation according to the left pulmonary artery. PLSVC is seen in the anterior aspect of the left pulmonary artery, while the LACV is in the posterior aspect. The evaluation of the flow direction with echocardiography or velocity-encoded cine magnetic resonance imaging is another way to make differential diagnoses. The blood flows in the caudocranial direction in the LACV while it flows craniocaudal direction in the PLSVC. However, the bidirectional flow could be seen in the levoatriocardinal vein –31.Moreover, the caudocranial flow may be observed in PLSVC when there is atresia or stenosis of the CS ostium. Identification of accompanying CA may also help in the differential diagnosis. If LOLs without ASD are present, LACV should be considered in the differential diagnosis, firstly. It is hypothesized that, in the presence of in utero LOLs such as mitral stenosis, collaterals between pulmonary and systemic circulations cannot regress due to increased pressure in the left atrium and remain as LACV in the postnatal period. However, in the presence of complex CA, the diagnosis of PLSVC should be considered mainly –31.LACV could be isolated without any CA, like PLSVC. Nevertheless, the frequency of this probability is very low for LACV compared to PLSVC. Additionally, they may be seen together, and the LACV may drain into PLSVC , 30.The pericardiophrenic veins (PCPV) are responsible for pericardial and diaphragmatic venous drainage. They lie along the lateral border of the heart and mediastinum, accompany pericardiophrenic arteries/phrenic nerve and drain into the internal thoracic, superior intercostal, or BCV. Due to the connection with inferior phrenic veins, dilated PCPVs could be observed as a collateral pathway in cases of SVC or IVC occlusion. Besides, they can serve as a collateral route via portosystemic shunting in portal hypertension Fig.  32–34].–34.32–34In the case of catheters located at the left paramediastinal region, the left PCPV is one of the possible differential diagnoses. In posteroanterior chest X-ray, left PCPV has a lateral course along the left heart border, while PLSVC turns medially near the left atrium. Although they both are located in the middle mediastinum and connected with left brachiocephalic vein cranially, their caudal courses differ in CT imaging. While the caudal end of PLSVC is either the coronary sinus or the left atrium, the left pericardiophrenic vein moves toward the diaphragm lateral to the heart when it is followed from top to bottom , 36.The left superior intercostal vein (L-SICV) drains the blood from the second, third, and fourth left intercostal veins into RSVC through the hemiazygos/azygos venous systems. Ordinarily, it can be seen as a small aortic nipple (1.4–5%) on the chest radiograph and is indistinguishable in CT. If its diameter exceeds 4.5 mm, it should be considered as abnormal. In the case of occlusion of SVC at the distal level of the azygos vein, the connection between SVC and IVC becomes possible with the dilation of L-SICV and other collateral vessels Fig.  9, 37, , 37, 38.Furthermore, L-SICV may dilate in congenital conditions such as hypoplasia of LBCV, and diseases leading to volume overload such as congestive heart failure. In such cases, L-SICV might be confused with PLSVC. However, knowing their courses and drainage sites will facilitate the diagnosis , 37, 38.Aberrant left brachiocephalic vein (ALBV) is a rare anomaly (≈ 1%) and is often associated with CAs, such as TOF, septal defects, and right atrial isomerism. Ordinarily, the LBCV passes through the anterior of the arcus aorta and connects with the right BCV. In the presence of an aberrant course, the LBCV begins with the junction of the left subclavian and jugular veins, moves inferiorly along the left side of the mediastinum, and joins to the right BCV passing behind the ascending aorta or esophagus. Retroesophageal ALBV is a more rare variation Fig.  10, 11,,11, 39.Vascular structures located on the left side of the mediastinum in patients with the history of cardiac surgery performed for complex CA may also be included in the differential list of PLSVC. The differential diagnosis could be made by knowing the performed surgery and demonstrating the drainage site of the vessel. Bicaval Glenn shunt, the left-sided Blalock-Taussig (BT) shunt, and collateral vessels after Fontane surgery are possible differentials Fig. .Fig. 23Bicaval Glenn shunt is an anastomosis of both SVCs to pulmonary arteries in the presence of PLSVC. The Glenn shunt allows the direct drainage of venous blood into the pulmonary arteries via bypassing the right heart chambers , 41.BT shunt is one of the surgical methods for complex CA. In this procedure, the connection of the subclavian artery and the pulmonary artery is enabled via the graft placement , 42.In Fontan surgery, the SVC and IVC are anastomosed to the pulmonary artery. After surgery, collaterals, which may be seen as large vessels on the left side of the mediastinum, may develop and may be confused with PLSVC .In conclusion, PLSVC is the most common thoracic venous anomaly known to be mostly asymptomatic. However, contrary to common misconception, it may cause a number of clinically significant symptoms, even in a heart with normal anatomy. Likewise, it may significantly affect the proper approaches to heart transplantations, effective surgical treatments for complex cardiac anomalies, and ablative procedures for cardiac arrhythmias. Thus, it should be recognized correctly and reported explicitly in radiological reports, even when it is an incidental finding. Besides, it is important to be aware of differential diagnoses of PLSVC and their radiological features to correctly interpret the vascular structures on the left side of the mediastinum.


Extensive milling processes have deprived wheat flour from essential nutrients. The objective of the current study was to assess the nutritive quality of commercial wheat flour (soft flour (SF)) through analyses of proximate composition and functional properties as well as quantification of benzoyl peroxide  by comparing the results with whole wheat flour .Triticum aestivum L.; Inqulab 91) crop grown in the experimental field of University of Agriculture, Faisalabad, under optimized field conditions without any fertilizers and insecticides. Functional properties  and proximate composition  were determined and compared for all the samples. Benzoyl peroxide (BPO) and Benzoic Acid (BA) quantification was performed through High Performance Liquid Chromatography. Finally dietary intake was estimated for BPO and BA.The samples included commercial SF purchased from the local supplier of different flour mills (who use BPO as additive) and a control sample without additives was prepared by grinding the seeds harvested from wheat  showed dissociation of BPO into BA soon after the exposure. Observed BA range (13.77 mg/g after 16 h) in SF and exposure level assessment (44.3 ± 1.36 mg/kg/BW) showed higher intake of BA on the consumption of SF. The results revealed the superiority of WF over SF in nutritive qualities as well as free of toxicants such as BA. Triticum aestivum L.) is a principal cereal consumed worldwide in different forms as a major food source. During milling process the grains are milled to obtain flour which is pale yellow in color. The bakery products it yields are less commercially acceptable, therefore flour is bleached for different bakery items. When this flour is stored, natural maturation or aging takes place, where carotenoids undergo oxidation and improves rheological properties of the dough such as texture (Wheat ( texture . Lutein  texture .Wheat milling industries utilize oxidizing agents such as benzoyl peroxide (BPO) to accelerate the process of maturation and dough improvement. These agents oxidize sulf-hydryl groups in flour gluten protein that yields increased stickiness of the flour and makes its appearance as soft flour (SF) . These oBeside addition of BPO, the SF is processed several times compared to the WF to achieve the fiber free fine particles for the improved texture of the bakery products. During this processing structure, sensory qualities, protein contents, fiber contents and nutritional components get severely affected. Antioxidants in WF, which are present in the germ and bran , are mosn = 4, SF) purchased from the local supplier from different flour mills (with additives) and a control sample  without additives was prepared by mechanical grinding the Triticum aestivum L. seeds (Inqulab 19), harvested from the crop grown on the experimental field of University of Agriculture, Faisalabad, under optimized field conditions  without any fertilizer and insecticide. The variety of wheat seeds and growth conditions were kept same as used by flour mills to generate SF, to avoid ambiguity in results. The WF flour sample was passed through sieve (75 µm size) before packing them into air-tight plastic containers. The size was in agreement with the particle size of SF purchased from flour mills.Test samples included commercial SF (g) is the weight of the sample and volume (mL) is the volume of the material. Water absorption capacity and oil absorption capacity (WAC and OAC respectively) were determined through the method described by V is the volume of water (mL). Similar procedure was repeated for OAC determination using soybean oil (Sp. Gravity 0.9092) as the absorbing agent. Emulsifying stability and emulsifying activity (ES and EA respectively) were determined by the method described by Bulk density of flour was determined as described previously  by folloW1 is weight of Gooch crucible before ashing, W2 is weight of Gooch Crucible containing asbestos and ash and W is weight of the dried material taken for the test. The fat content of wheat was determined using the method of Association of Official Analytical chemists  as well as conventional methods to get reliable results. These properties included moisture content (MC), ash contents (AC), crude protein (CP), gluten and starch contents. Other properties such as crude fiber and fat (CFF) were determined through solvent extractions. For crude fiber about 2.5–3 g of sample was transferred to Soxhlet and extracted with petroleum ether. The air dried extracted sample was transferred to one L conical flask. Whole boiling dilute sulfuric acid (1.25% w/v) was transferred to the flask containing sample and immediately connected with a water cooled reflux condenser and heat it so that the contents start boiling in 1 min. Few drops of octanol were added to avoid bumping and risk of foaming. The flask was continuously shaken and boiled for 30 min. Flask is then removed and filtered through fine linen (18 threads to a cm) placed in a funnel and washed with boiling water until no longer acid to litmus is achieved. The residue on linen was then washed with the 200 mL of boiling NaOH (1.25% w/v). Immediately the flask was connected to the reflux condenser and boiled for 30 min. The residue was then filtered through filtering cloth and residues were thoroughly washed with boiling water. The residues were transferred to Gooch crucible having thin compact layer of ignited asbestos. Finally wash the residues with 15 mL ethyl alcohol. The Gooch crucible was dried in air oven at 105 ± 2 °C in an air oven until constant weight is achieved. The crucible was weighed and placed in a muffle furnace to burnt all carbonaceous matter. Crucible was then cooled and weighed again. Crude fiber was then calculated by the following formula; chemists  with petSince the bulk density  varies with MC, therefore, it was determined following procedure described in ASABE standard S358.2 . During NIR Omega G Analyser  was employed to analyze different parameters  of the flour and grain samples. The spectral transmissions range was 700–1,100 nm with 5 nm scan increment, measured at controlled room conditions of 24 ± 1 °C, RH 34 ± 2%.−1 and spectra generated were interpreted on the basis overtones of different functional groups in the product. Resolution of spectrometer was 4 cm−1  with maximum scan time kept at 5 s. MIR spectra were recorded at Opus 6.0 Burker Software using Attenuated Total Reflectance (ATR) unit. The reference spectrum (empty sample bottle) was utilized as background measurement before loading in sample’s spectra. About 8–10 g of sample was added in the sample bottle to generate the spectra in diffused reflectance mode. Three spectra per sample were recorded by rotating the sample bottle at 120°. The measurements were carried out under the controlled conditions of temperature 24 ± 1 °C, RH 34 ± 2%. Each spectra were the average of three scans per object.The FTIR transmission spectra were recorded at Burker-TENSOR 37 FTIR spectrometer with Michelson interferometer. Working range of the spectrometer was 4,000–12,000 cm2) to attain the homogeneous blend and kept in dark. The bleaching reaction (performed at room temperature) was monitored after every hour. The product of reaction was extracted every hour for a period of 8 h, finally a sample was taken at 12 h and 16 h. Then samples were analyzed by High Performance Liquid Chromatography (HPLC) and compared with the commercial standards and calibration graph for the quantification. Associated peaks (matching with the standard) were considered for the generation of results.Benzoyl peroxide and BA were quantified in wheat flour as described by the method of . Whole wStandard stock solution (SS) was generated by dissolving pure BPO (60 mg/L) in diethyl ether (100% purity). Working standards were obtained by diluting SS with appropriate volume of diethyl ether. For BA stock solution, pure compound (100 mg) was dissolved in 100 mL of methanol and working standards were prepared by diluting the stock solution. Calibration curve was then generated by plotting the absorbance value against concentration.The standard procedure was carried out (at room temperature) in a flask with grinding stopper. About 100 mL of diethyl ether was added to 50 g of flour . This mixture was shaken vigorously on a magnetic stirrer for 10 min and left to settle for 15 min. Upper layer of this solution (containing the products of reaction) was withdrawn through the pipette and transferred into Falcon polypropylene tube (10 mL) and held into ice until HPLC analysis.The supernatant was analyzed by Waters 600 HPLC system at Inertsil ODS-80A column  equipped Inertsil ODS-3 guard column (10 × 4 mm i.d.) and Waters 2,996 Photodiode array detector. The detection wavelength was kept at 235 nm and column oven at 40 °C. For isocratic separation the conditions were as follows: Water (Solvent A), acetonitrile  and Benzoic acid (Solvent C); 55% B:45% A as mobile phase for 1 mL/min. The gradient conditions for analysis were as follows: Water-glacial acetic acid  (Solvent A), ACN-glacial acetic acid  (solvent B); 18% B (10 min) was increased to 60% B (11–15 min hold) at flow rate 1.2 mL/min and column temperature 35 °C.Xv = average daily amount (kg) of wheat flour consumed by a subject; Cv = concentration of BA (mg/kg) as determined through HPLC in particular wheat flour sample; Bw = Average body weight of the subject.The mean dietary intake for both BP and BA were estimated to determine the exposure rate. For this purpose, 200 subjects  were evaluated for their preferences of WF and SF as well as amount of daily intake through a questionnaire survey. The SF brands, which were reported to be consumed, were actually evaluated for the presence of BA in the current study. Further calculations were accomplished through the following equation:P < 0.01 and P < 0.05). The aim was to give the significant difference in the data sets from different flour samples which was not achievable through univariate or one-way ANOVA.All samples were analyzed in triplicate , therefore standard error of mean (SEM) was applied using the Statistixl 1.9 Add-in package within Excel 2007. Two-way analysis of variance (ANOVA) was conducted, followed by post-hoc Tukey tests that separated the treatment into groups  were compared for their functional properties, emulsifying properties, foaming capacity, gelation capacity, proximate composition and benzoyl peroxide composition. Finally, the exposure of benzoyl peroxide was compared with the daily intake capacity to observe the exposure of consumers when they are consuming SF or WF. The samples of SF were collected from the flour mills which were preferably consumed by the subjects. WF samples were collected from same seed variety (Inqulab 19) grown under similar conditions as wheat of SF. Detailed results are as under.The oil absorption capacity OAC;  showed tP < 0.01) was observed when values were compared statistically with those of WF.The results revealed that emulsifying Activity (EA) was higher for WF (43.7%), whereas, stability (ES) is higher for all SF samples (<42%). EA and ES of the WF and SF may also vary due to the milling process. The emulsifying properties varied inversely, which means that WF had highest EA and lowest ES. Foaming Capacity (FC) and Foaming stability (FS) collectively form the foaming properties of any flour. Both of these properties are directly proportional to one another, which were observed to be higher for WF (12.9% FC and 1.94% FS). FC and FS of WF is more (<12% and <1% respectively) compared to all SF samples (>9% and >1% respectively). A highly significant difference (P < 0.001 when datasets were compared with the WF dataset). It can also be observed that both considered parameters for gelation capacity are directly related to each other such that increase in one also showed increase in the other.Gelation capacity (including gelatinization temperature GnT and least Gelatinization concentration LGC) are attributed and controlled by the balance between hydrophilic interactions and repulsive electrostatic interactions between the water/oil and proteins . ResultsP < 0.01) when compared with the WF dataset. This showed that quality of flour was deteriorated while processing and refining.The proximate composition  of all the flour samples is as summarized in the Benzoyl peroxide and BA were determined simultaneously using gradient analysis . The retBenzoyl peroxide is a free radical initiator and it causes the oxidation of carotenoids by free radical mechanism. The process  leads toEstimation of dietary exposure of BA through just wheat flour consumption was estimated . Since fHigh Performance Liquid Chromatography was used for quantification of BP in both white and WF samples. BA was also determined because BP was decomposed into benzoic acid within limited days . A reseaWhole wheat flour and SF were compared for their properties such as functional properties, emulsifying properties, foaming capacity, gelation capacity, proximate composition and benzoyl peroxide composition. The comparison was developed to aware and understand the choice of flour in daily meals and their potential associated risks.The oil absorption capacity (OAC) of the flour varies with the intrinsic properties such as amino acid composition, protein conformation, hydrophilic-hydrophobic balance of amino acids, steric factors as well as lipid and carbohydrate composition of a flour sample . “ResultSwelling, another important functional property referred to as spontaneous uptake of water by protein matrix, is indirectly related to WHC. Since SF is lower in WHC therefore its swelling capacity is more (>18 compared to 17.8 in case of WF), which ensures longer life of bakery items. Water retention is very important for protein functionality which determines the cationic, non-ionic and anionic polar sites of the protein molecules on the flour surface . Lower WThe emulsifying properties vary inversely with the insoluble protein fractions and polysaccharides . The unfProtein gelation is very important in several vegetables and other food items. The effective overlapping of the functional groups between adjacent protein moieties is very important for the gel network formation. Higher gelation capacity (Gelatinization temperature) for WF indicates that this may not be a good choice in bakery items.P < 0.001) with the WF dataset. This shows that quality of SF was deteriorated during processing and refining.The flour at the beginning of the process comes from the endosperm, which is rich in starch and as it reaches the end of grinding, flour comes from periphery which is rich in ash, pentosan and protein. During the milling process, the seeds pass through heavy grinders to attain fine powdered flour. This flour is further sieved and final product, obtained after series of sieving and treatment with BP, is packed and sold as commercial white flour used for bakery items. Most of the components such as crude protein, gluten, damaged starch datasets showed highly significant difference (Flour mills consume benzoyle peroxide (BP) to improve the appearance and white color of flour. BP is a free radical initiator and therefore, it causes the oxidation of carotenoids by free radical mechanism. The process  leads toWheat flour is an unavoidable commodity. This shows that white SF as produced out of milling process are contributors of excessive amount of toxic benzoic acid (BA) in the consumers’ body. Results of current study indicated very high amount of BA entering in human body upon consumption of SF. In China standard limit of BPO is not exceed 60 mg/kg; the maximum content of BP in wheat flour is 80 mg/kg in the US, in Japan 300 mg/kg, and also 50 mg/kg in United Kingdom. In China, permissible amount of BPO are 0.045, 0.05 0.06 g/kg according to standards of food additives regulation . AccordiWheat flour is one of the most important ingredients of food being consumed most frequently. To improve the baking quality, SF is often used instead of WF. Increased demands of fine texture and bleached color has led flour millers to add enhanced concentration of BPO and extensive milling along with sieving. Extensive milling and increased BPO reduce the nutritive value of SF and enrich it with the toxicant such as BA (as degradation product of BPO). Among various parameters of flour quality, protein and ash content in WF sample was more than SF samples. Therefore, there is need to improve wheat flour quality being sold in the market by limiting the rate of BPO added as bleaching agent as well milling process should be improved. HPLC analyses effectively demonstrated the dissociation of BPO to BA, which means that BA in SF was due to BPO added as bleaching agent.10.7717/peerj.8788/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8788/supp-2Supplemental Information 2Click here for additional data file.