A detailed review on advanced oxidation process in treatment of wastewater: Mechanism, challenges and future outlook.

The presence of several contaminants in waterbodies raises global pollution and creates major risks to mankind, wildlife, as well as other living organisms. Development of an effective, feasible, cost-effective and eco-friendly approach for treating wastewater that is discharged from various industries is important for bringing down the deposition of contaminants into environment. Advanced oxidation process is an efficient technique for treating wastewater owing to its advantages such as high oxidation efficacy and does not produce any secondary pollutants. Advanced oxidation process can be performed through various methods such as ozone, Fenton, electrochemical, photolysis, sonolysis, etc. These methods have been widely utilized for degradation of emerging pollutants that cannot be destroyed using conventional approaches. This review focuses on wastewater treatment using advanced oxidation process. A brief discussion on mechanism involved is provided. In addition, various types of advanced oxidation process and their mechanism are explained in detail. Challenges faced during wastewater treatment process using oxidation, electrochemical, Fenton, photocatalysis and sonolysis are discussed elaborately. Advanced oxidation process can be viewed as potential approach for treating wastewater with certain modifications and solving challenges.

Hereditary neuropathies: Genetics and utility of nerve biopsy.

Peripheral neuropathy is one of the most common neurological conditions of the nervous system. Hereditary neuropathies (HNs) form an important group with varying degrees of severity, causing a significant disease burden. Accurate diagnosis is essential for management, counseling, and preventing unnecessary extended workups for acquired etiologies and inappropriate treatment. Several hereditary neuropathies have characteristic or diagnostic histologic findings; however, in the era of molecular diagnostics, the role of nerve biopsy in the diagnosis of hereditary neuropathy has reduced significantly. Nevertheless, in sporadic cases, cases without a clear family history, clinical mimics, cases with rare mutations, and genetic variants of unknown significance, a nerve biopsy can confirm the diagnosis, provide an unexpected diagnosis, or direct a targeted molecular testing. HN may be non-syndromic, affecting predominantly the peripheral nervous system or syndromic where it is a part of more widespread neurological or multisystem involvement. This review summarizes the microscopic pathological features in a nerve biopsy in some of the more commonly encountered inherited peripheral neuropathies highlighting their utility in selected cases.

Key players in regulatory RNA realm of bacteria.

Precise regulation of gene expression is crucial for living cells to adapt for survival in diverse environmental conditions. Among the common cellular regulatory mechanisms, RNA-based regulators play a key role in all domains of life. Discovery of regulatory RNAs have made a paradigm shift in molecular biology as many regulatory functions of RNA have been identified beyond its canonical roles as messenger, ribosomal and transfer RNA. In the complex regulatory RNA network, riboswitches, small RNAs, and RNA thermometers can be identified as some of the key players. Herein, we review the discovery, mechanism, and potential therapeutic use of these classes of regulatory RNAs mainly found in bacteria. Being highly adaptive organisms that inhabit a broad range of ecological niches, bacteria have adopted tight and rapid-responding gene regulation mechanisms. This review aims to highlight how bacteria utilize versatile RNA structures and sequences to build a sophisticated gene regulation network.

Carfilzomib: A Promising Proteasome Inhibitor for the Treatment of Relapsed and Refractory Multiple Myeloma.

The proteasome is crucial for the degradation of intracellular proteins and plays an important role in mediating a number of cell survival and progression events by controlling the levels of key regulatory proteins such as cyclins and caspases in both normal and tumor cells. However, compared to normal cells, cancer cells are more dependent on the ubiquitin proteasome pathway (UPP) due to the accumulation of proteins in response to uncontrolled gene transcription, allowing proteasome to become a potent therapeutic target for human cancers such as multiple myeloma (MM). Up to date, three proteasome inhibitors namely bortezomib (2003), carfilzomib (2012) and ixazomib (2015) have been approved by the US Food and Drug Administration (FDA) for the treatment of patients with relapsed and/or refractory MM. This review mainly focuses on the biochemical properties, mechanism of action, toxicity profile and pivotal clinical trials related to carfilzomib, a second-generation proteasome inhibitor that binds irreversibly with proteasome to overcome the major toxicities and resistance associated with bortezomib.

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

BACKGROUND: Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. METHODOLOGY: To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. PRINCIPAL FINDINGS: Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 ß-cells by pentamidine may be secondary to pentamidine-mediated activation of ß-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

Exposure to the neurotoxin 3-nitropropionic acid in neuronal cells induces unique histone acetylation pattern: Implications for neurodegeneration.

Mitochondrial dysfunction is critical for neurodegeneration in movement disorders. Neurotoxicological models recapitulating movement disorder involve mitochondrial damage including inhibition of mitochondrial complexes. Previously, we demonstrated that neurotoxic models of Parkinson's disease and Manganism showed distinct morphological, electrophysiological and molecular profile indicating disease-specific characteristics. In a recent study, we demonstrated that the transcriptomic changes triggered by the neurotoxic mitochondrial complex II inhibitor 3-nitropropionic acid (3-NPA), was significantly different from the profile induced by the neurotoxic mitochondrial complex I inhibitor 1-methyl-4- phenylpyridinium (MPP+) and mitochondrial toxin Manganese (Mn). Among the plausible pathways, we surmised that epigenetic mechanisms could contribute to 3-NPA specific transcriptomic profile. To address this, we assessed global and individual lys-specific acetylation profile of Histone H3 and H4 in the 3-NPA neuronal cell model. Our data revealed histone acetylation profile unique to the 3-NPA model that was not noted in the MPP+ and Mn models. Among the individual lys, Histone H3K56 showed robust dose and time-dependent hyperacetylation in the 3-NPA model. Chromatin Immunoprecipitation-sequencing (ChIP-seq) revealed that acetylated H3K56 was associated with 13072 chromatin sites, which showed increased occupancy in the transcription start site-promoter site. Acetylated histone H3K56 was associated with 1747 up-regulated and 263 down-regulated genes in the 3-NPA model, which included many up-regulated autophagy and mitophagy genes. Western analysis validated the involvement of PINK1-Parkin dependent mitophagy in the 3-NPA model. We propose that 3-NPA specific chromatin dynamics could contribute to the unique transcriptomic profile with implications for movement disorders.

Clinical diversity and treatment approaches to blastic plasmacytoid dendritic cell neoplasm: a retrospective multicentre study.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive type of haematologic precursor malignancy primarily often manifesting in the skin. We sought to provide a thorough clinical characterization and report our experience on therapeutic approaches to BPDCN. METHODS: In the present multicentric retrospective study, we collected all BPDCN cases occurring between 05/1999 and 03/2018 in 10 secondary care centres of the German-Swiss-Austrian cutaneous lymphoma working group. RESULTS: A total of 37 BPDCN cases were identified and included. Almost 90% of the patients had systemic manifestations (bone marrow, lymph nodes, peripheral blood) in addition to skin involvement. The latter presented with various types of cutaneous lesions: nodular (in more than 2/3) and bruise-like (in 1/3) skin lesions, but also maculopapular exanthema (in circa 1/6). Therapeutically, 22 patients received diverse combinations of chemotherapeutic regimens and/or radiotherapy. Despite initial responses, all of them ultimately relapsed and died from progressive disease. Eleven patients underwent haematopoietic stem cell transplantation (HSCT; autologous HSCT n = 3, allo-HSCT n = 8). The mortality rate among HSCT patients was only 33.33% with a median survival time of 60.5 months. CONCLUSION: Our study demonstrates the clinical diversity of cutaneous BPDCN manifestations and the positive development observed after the introduction of HSCT.

Mapping the protein phosphorylation sites in human mitochondrial complex I (NADH: Ubiquinone oxidoreductase): A bioinformatics study with implications for brain aging and neurodegeneration.

In eukaryotes, mitochondrial complex I (NADH: ubiquinone oxidoreductase; CI) is central to oxidative phosphorylation (OXPHOS). Mammalian CI is a 45 subunit complex that forms supercomplexes with other OXPHOS complexes. Since CI defects are associated with aging and neurodegeneration, it is pertinent to understand its structure-function relationship. Although genetic mutations could lower CI activity causing mitochondrial dysfunction in several pathologies, post-translational modifications (PTMs) have emerged as a key mechanism contributing to altered CI activity. Among non-oxidative PTMs, protein phosphorylation is the most intricate regulatory mechanism controlling CI structure and function during normal physiology, aging and neurodegeneration. To comprehend this, we carried out a comprehensive bioinformatics analysis of protein phosphorylation of human CI subunits using software-based prediction of phosphorylation (phospho) sites and associated kinases. Phosphorylation was higher among core subunits and active domains of the complex. Among the subunits, NDUFS1 displayed significantly higher number as well as percent phospho sites compared to others. Analysis of the subunits containing iron-sulfur (Fe-S) cluster, NADH and FMN binding sites and quinone binding sites indicated the presence of phospho sites in close proximity to the binding sites of these cofactors with potential functional implications. Phosphoproteomics experiment in rat and human muscle mitochondria identified specific phospho sites in CI subunits, thereby validating the bioinformatic analysis. Molecular modeling of CI subunits indicated structural implications following phosphorylation. We surmise that protein phosphorylation, a transient and regulatory event could influence the structure-function relationship of CI thereby impinging on bioenergetics and ultimately contributing to aging and neurodegeneration.

Active dendritic integration and mixed neocortical network representations during an adaptive sensing behavior.

Animals strategically scan the environment to form an accurate perception of their surroundings. Here we investigated the neuronal representations that mediate this behavior. Ca2+ imaging and selective optogenetic manipulation during an active sensing task reveals that layer 5 pyramidal neurons in the vibrissae cortex produce a diverse and distributed representation that is required for mice to adapt their whisking motor strategy to changing sensory cues. The optogenetic perturbation degraded single-neuron selectivity and network population encoding through a selective inhibition of active dendritic integration. Together the data indicate that active dendritic integration in pyramidal neurons produces a nonlinearly mixed network representation of joint sensorimotor parameters that is used to transform sensory information into motor commands during adaptive behavior. The prevalence of the layer 5 cortical circuit motif suggests that this is a general circuit computation.

Variations in HLA-B cell surface expression, half-life and extracellular antigen receptivity.

The highly polymorphic human leukocyte antigen (HLA) class I molecules present peptide antigens to CD8+ T cells, inducing immunity against infections and cancers. Quality control mediated by peptide loading complex (PLC) components is expected to ensure the cell surface expression of stable peptide-HLA class I complexes. This is exemplified by HLA-B*08:01 in primary human lymphocytes, with both expression level and half-life at the high end of the measured HLA-B expression and stability hierarchies. Conversely, low expression on lymphocytes is measured for three HLA-B allotypes that bind peptides with proline at position 2, which are disfavored by the transporter associated with antigen processing. Surprisingly, these lymphocyte-specific expression and stability differences become reversed or altered in monocytes, which display larger intracellular pools of HLA class I than lymphocytes. Together, the findings indicate that allele and cell-dependent variations in antigen acquisition pathways influence HLA-B surface expression levels, half-lives and receptivity to exogenous antigens.

Preparation, characterization, drug release and computational modelling studies of antibiotics loaded amorphous chitin nanoparticles.

We present a computational investigation of binding affinity of different types of drugs with chitin nanocarriers. Understanding the chitn polymer-drug interaction is important to design and optimize the chitin based drug delivery systems. The binding affinity of three different types of anti-bacterial drugs Ethionamide (ETA) Methacycline (MET) and Rifampicin (RIF) with amorphous chitin nanoparticles (AC-NPs) were studied by integrating computational and experimental techniques. The binding energies (BE) of hydrophobic ETA, hydrophilic MET and hydrophobic RIF were -7.3kcal/mol, -5.1kcal/mol and -8.1kcal/mol respectively, with respect to AC-NPs, using molecular docking studies. This theoretical result was in good correlation with the experimental studies of AC-drug loading and drug entrapment efficiencies of MET (3.5±0.1 and 25± 2%), ETA (5.6±0.02 and 45±4%) and RIF (8.9±0.20 and 53±5%) drugs respectively. Stability studies of the drug encapsulated nanoparticles showed stable values of size, zeta and polydispersity index at 6°C temperature. The correlation between computational BE and experimental drug entrapment efficiencies of RIF, ETA and MET drugs with four AC-NPs strands were 0.999 respectively, while that of the drug loading efficiencies were 0.854 respectively. Further, the molecular docking results predict the atomic level details derived from the electrostatic, hydrogen bonding and hydrophobic interactions of the drug and nanoparticle for its encapsulation and loading in the chitin-based host-guest nanosystems. The present results thus revealed the drug loading and drug delivery insights and has the potential of reducing the time and cost of processing new antibiotic drug delivery nanosystem optimization, development and discovery.

Detection of Dysferlin Gene Pathogenic Variants in the Indian Population in Patients Predicted to have a Dysferlinopathy Using a Blood-based Monocyte Assay and Clinical Algorithm: A Model for Accurate and Cost-effective Diagnosis.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) is the most common adult-onset class of muscular dystrophies in India, but a majority of suspected LGMDs in India remain unclassified to the genetic subtype level. The next-generation sequencing (NGS)-based approaches have allowed molecular characterization and subtype diagnosis in a majority of these patients in India. MATERIALS AND METHODS: (I) To select probable dysferlinopathy (LGMD2B) cases from other LGMD subtypes using two screening methods (i) to determine the status of dysferlin protein expression in blood (peripheral blood mononuclear cell) by monocyte assay (ii) using a predictive algorithm called automated LGMD diagnostic assistant (ALDA) to obtain possible LGMD subtypes based on clinical symptoms. (II) Identification of gene pathogenic variants by NGS for 34 genes associated with LGMD or LGMD like muscular dystrophies, in cases showing: absence of dysferlin protein by the monocyte assay and/or a typical dysferlinopathy phenotype, with medium to high predictive scores using the ALDA tool. RESULTS: Out of the 125 patients screened by NGS, 96 were confirmed with two dysferlin variants, of which 84 were homozygous. Single dysferlin pathogenic variants were seen in 4 patients, whereas 25 showed no variants in the dysferlin gene. CONCLUSION: In this study, 98.2% of patients with absence of the dysferlin protein showed one or more variants in the dysferlin gene and hence has a high predictive significance in diagnosing dysferlinopathies. However, collection of blood samples from all over India for protein analysis is expensive. Our analysis shows that the use of the "ALDA tool" could be a cost-effective alternative method. Identification of dysferlin pathogenic variants by NGS is the ultimate method for diagnosing dysferlinopathies though follow-up with the monocyte assay can be useful to understand the phenotype in relation to the dysferlin protein expression and also be a useful biomarker for future clinical trials.

Family with Ehlers-Danlos syndrome (combined classic and vascular type) with rare presentation of progressive myopathy and unusual association of severe facial and trigeminal motor weakness.

We report the clinical, radiological, biochemical, muscle histology, and electron microscopic features of two members of a family with combined Ehlers-Danlos syndrome (EDS) [classic and vascular type] and progressive myopathy as the primary manifestation. A 35-year old lady presented with severe gluteal and thigh muscle pain and easy fatigability for 5 years. She developed weakness and wasting of pelvic and pectoral girdles and thighs for 3 years and severe neck flexor and truncal weakness for 6 months. She had a history of recurrent jaw dislocation, easy bruising with hyperpigmentation, hyperextensibility of joints, translucent skin, and papyraceous scars. She had high myopia with astigmatism. She had wasting of temporalis, masseters, sternocleidomastoids and trapezius. There was moderate weakness of temporalis, masseters, and facial muscles. Muscle power was Medical Research Council (MRC) grade 4 at shoulders and arms, and grade 3+ at pelvis and thighs. Serum homocysteine level was normal, and creatine kinase (CK) was 275 IU. Two dimensional echocardiogram (2D Echo) showed myxomatous degeneration of mitral valves. Electromyography (EMG) was suggestive of a myopathic pattern. Muscle magnetic resonance imaging (MR) revealed severe fatty infiltration of paraspinal muscles, gluteus maximus and medius, quadriceps, hamstrings, and gastrocnemius. Electron microscopy showed an occasional distorted fibril with mild increase in oxytalan fibers and variation in thickness of blood vessel basement membrane. Her 15-year old daughter had exertion-induced myalgias, right hemifacial hypoplasia, myopia, hyperextensible joints, hyperelastic skin, and neck muscle weakness. However, her CK and 2D Echo were normal. This report presents the rare combination of classic and vascular type of EDS primarily presenting as muscle weakness and associated with facial and trigeminal motor weakness.

Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB).

Pentamidine is an effective trypanocidal drug used against stage 1 Human African Trypanosomiasis (HAT). At the blood-brain barrier (BBB), it accumulates inside the endothelial cells but has limited entry into the brain. This study examined transporters involved in pentamidine transport at the human and mouse BBB using hCMEC/D3 and bEnd.3 cell lines, respectively. Results revealed that both cell lines expressed the organic cation transporters (OCT1, OCT2 and OCT3), however, P-gp was only expressed in hCMEC/D3 cells. Polarised expression of OCT1 was also observed. Functional assays found that ATP depletion significantly increased [3H]pentamidine accumulation in hCMEC/D3 cells (***p<0.001) but not in bEnd.3 cells. Incubation with unlabelled pentamidine significantly decreased accumulation in hCMEC/D3 and bEnd.3 cells after 120 minutes (***p<0.001). Treating both cell lines with haloperidol and amantadine also decreased [3H]pentamidine accumulation significantly (***p<0.001 and **p<0.01 respectively). However, prazosin treatment decreased [3H]pentamidine accumulation only in hCMEC/D3 cells (*p<0.05), and not bEnd.3 cells. Furthermore, the presence of OCTN, MATE, PMAT, ENT or CNT inhibitors/substrates had no significant effect on the accumulation of [3H]pentamidine in both cell lines. From the data, we conclude that pentamidine interacts with multiple transporters, is taken into brain endothelial cells by OCT1 transporter and is extruded into the blood by ATP-dependent mechanisms. These interactions along with the predominant presence of OCT1 in the luminal membrane of the BBB contribute to the limited entry of pentamidine into the brain. This information is of key importance to the development of pentamidine based combination therapies which could be used to treat CNS stage HAT by improving CNS delivery, efficacy against trypanosomes and safety profile of pentamidine.

Characterization of age-dependent changes in the striatum: Response to the mitochondrial toxin 3-nitropropionic acid.

Neurodegenerative phenomena are associated with mitochondrial dysfunction and this could be exacerbated by aging. Age-dependence of mitochondrial response to toxins could help understand these mechanisms and evolve novel therapeutics. 3-Nitropropionic acid (3-NPA) is a mitochondrial toxin that induces neurotoxicity in the striatum via inhibition of complex II. We investigated the age-related events that contribute to 3-NPA toxicity. 3-NPA induced neuronal death, oxidative stress and altered mitochondrial structure in neuronal cells. 3-NPA injection in vivo caused motor impairment, mitochondrial dysfunction and oxidative damage with different trend in young and adult mice. To understand the age-dependent mechanisms, we carried out proteomic analysis of the striatal protein extract from young mice (control: YC vs. 3-NPA treated: YT) and adult mice (control: AC vs. 3-NPA treated: AT). Among the 3752 identified proteins, 33 differentially expressed proteins (mitochondrial, synaptic and microsomal proteins) were unique either to YT or AT. Interestingly, comparison of the proteomic profile in AC and YC indicated that 161 proteins (linked with cytoskeletal structure, neuronal development, axogenesis, protein transport, cell adhesion and synaptic function) were down-regulated in AC compared to YC. We surmise that aging contributes to the cellular and molecular architecture in the mouse striatum with implications for neurodegeneration.

Advancement in contemporary diagnostic and therapeutic approaches for rheumatoid arthritis.

This review is intended to provide a summary of the pathogenesis, diagnosis and therapies for rheumatoid arthritis. Rheumatoid arthritis (RA) is a common form of inflammatory autoimmune disease with unknown aetiology. Bone degradation, cartilage and synovial destruction are three major pathways of RA pathology. Sentinel cells includes dendritic cells, macrophages and mast cells bound with the auto antigens and initiate the inflammation of the joints. Those cells further activates the immune cells on synovial membrane by releasing inflammatory cytokines Interleukin 1, 6, 17, etc., Diagnosis of this disease is a combinational approach comprises radiological imaging, blood and serology markers assessment. The treatment of RA still remain inadequate due to the lack of knowledge in disease development. Non-steroidal anti-inflammatory drugs, disease modifying anti rheumatic drugs and corticosteroid are the commercial drugs to reduce pain, swelling and suppressing several disease factors. Arthroscopy will be an useful method while severe degradation of joint tissues. Gene therapy is a major advancement in RA. Suppressor gene locus of inflammatory mediators and matrix degrading enzymes were inserted into the affected area to reduce the disease progression. To overcome the issues aroused from those therapies like side effects and expenses, phytocompounds have been investigated and certain compounds are proved for their anti-arthritic potential. Furthermore certain complementary alternative therapies like yoga, acupuncture, massage therapy and tai chi have also been proved for their capability in RA treatment.

Hereditary inclusion body myopathy: A myopathy with unique topography of weakness, yet frequently misdiagnosed: Case series and review of literature.

BACKGROUND: Hereditary inclusion body myopathy (HIBM) continues to be underrecognized clinically despite a characteristic topography of weakness with total sparing of quadriceps muscles and patient being wheelchair bound. We report seven patients of HIBM from four families in North India. METHODS AND RESULTS: Seven patients from four different families were diagnosed to have HIBM. There was no consanguinity in any of the families. While one patient had two affected siblings, another had one affected siblings and the family history was noncontributory in two patients. Two of the siblings were available for examination and confirmed clinically to be suffering from HIBM. Among the seven patients, only one was still ambulatory at the time of diagnosis. DISCUSSION: This is the first case report of occurrence of HIBM in North Indian population. Despite its unique clinical presentation, HIBM is frequently misdiagnosed resulting in unnecessary diagnostic and therapeutic interventions. A high index of suspicion of this rare myopathy along with proper clinical examination may go a long way in accurate prognostication and management of these patients.

Tangier's disease: An uncommon cause of facial weakness and non-length dependent demyelinating neuropathy.

Tangier disease is an autosomal recessive disorder characterized by an abnormal accumulation of cholesterol esters in various organs secondary to adenotriphosphate binding cassette transporter A-1 (ABCA-1) transporter deficiency and disrupted reverse cholesterol transport. It causes neuropathy in half of the affected individuals. We present the clinical, electrophysiological, and histopathological findings in a middle aged gentleman of Tangier disease who was initially misdiagnosed leprosy and treated with antileprosy drugs. The presence of a demyelinating neuropathy on electrophysiology in a patient with predominant upper limb involvement and facial diplegia should raise the suspicion of Tangier disease. The characteristic lipid profile of Tangier disease was noted in this patient viz. extremely low high density lipoprotein (HDL), elevated triglyceride (TG), and reduced apolipoprotein A1. Estimation of serum lipids should form a part of routine evaluation in order to avoid misdiagnosis.