Within-group statistical parametric mapping (SPM) analysis showed similar cerebellar activation during imagination of standing and its real execution between the PD and control groups ; i.e., increases in regional cerebral blood flow (rCBF) were observed in the anterior cerebellar vermis during motor imagination and the posterior vermis during standing. A comparison between the groups showed that the motor execution of standing significantly activated the superior part of the posterior vermis (declive VI) and the paracentral sulcus region in the PD patients, while the prefrontal cortices were deactivated during standing (p<0.001 uncorrected). Correlation analysis within the PD group revealed that the postural rCBF increases in the cerebellar vermis (pyramis) were negatively correlated with putaminal [11C]CFT binding  and that the postural rCBF reductions in the orbitofrontal cortex were positively correlated with caudate [11C]CFT binding . These results suggest that while the neural circuits for postural imagery and execution are intact in PD, standing performance, which requires more recruitment of dopaminergic control, may result in compensatory overstimulation of the cerebellar vermis and paracentral foot area in PD patients. Hyperactivity in these areas along with mesocortical hypofunction may be pathophysiological aspects of postural control in PD patients. Hence, our findings would help understand the modifications observed within the neural networks in relationship with postural performance, and possible compensatory mechanisms in PD.It has been reported that the cerebellar vermis is equally involved in both motor imagery about axial movement and the actual execution of postural balance in healthy human subjects, but this finding is yet to be explored in Parkinson’s disease (PD). We therefore investigated the neuronal responses during observation of standing posture, imagination of standing and the assumption of an upright posture in ten drug-naïve PD patients using positron emission tomography (PET) with [ Parkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic cell attrition, and its symptoms are characteristic of impaired motor function including bradykinesia, tremor and balance and gait disturbance . While aOur previous positron emission tomography (PET) study showed significant postural activation in the cerebellar vermis during standing and hyperactivation in the prefrontal cortex during eye-closing or motor imaging of standing in healthy human subjects . PreviouAs shown in behavioral improvement using external sensory cues in PD , motor-rHere, we aimed to clarify the relation of cognitive function (motor imagery) with the load of standing posture and discuss the possibility of the additional pathophysiology of PD by examining dopamine dysfunction and neuronal activations in parallel using our PET system.This study was approved by the Ethics Committee of Hamamatsu Medical Center, and written informed consent was obtained from all participants prior to taking part. A total of 22 subjects: 12 healthy subjects  and 10 right-handed PD patients  participated in this study. Healthy subjects were recruited via in-house advertisement and screened for exclusion criteria: regular intake of medicines, a history of psychiatric or neurological diseases, and contra-indications to MRI and PET scanning . Patients with PD were recruited through hospitals and clinics related to Hamamatsu Medical Center and screened for the same exclusion criteria with one important additional criterion: a history of any kind of dopamine therapy. In order to exclude the possibility of dopaminergic drug effect on dopamine PET data and CBF responses either under the on-state or off-state condition, we recruited all drug-naïve PD patients rated as the Hoehn-Yahr stage II or less, who did not suffer from significant postural instability but bradykinesic foot movoment. The patients were assessed using the Unified Parkinson’s Disease Rating Scale (UPDRS) and Mini-Mental State Examination (MMSE) . L-Dopa The participants were allowed several attempts to complete the tasks given in the PET scans prior to the measurement. First in the supine condition, we used a computer screen placed 1 meter before each subject, whilst they were lying on the scanner’s couch. The subjects were instructed to stare at a marker of a human silhouette that was standing in front of the door of our PET room displayed on the computer screen  was used with 3-dimensional mode sampling to determine the areas of the striatum in which to set the regions of interest (ROI). The MRI measures and a mobile PET gantry allowed us to reconstruct the PET images parallel to the intercommissural (ACPC) line without reslicing. Using this approach, we were able to allocate ROI on the target regions of the original PET images. .68Ge/68Ga source with the subject’s head fixed by a radiosurgery-purpose thermoplastic facemask, a 60-s emission scan was acquired from the time when the radiotracer first entered the cerebral circulation after an intravenous bolus injection of 300 MBq of H215O had been administered. 2-ß-carbomethoxy-3ß-(4-fluorophenyl) tropane ([11C]CFT). The detail was described elsewhere [11C]CFT injection and analyzed using thin-layer chromatography and a storage-phospher-screen bioimaging analyzer . The free metabolite-corrected plasma activities were fitted to a sum of three exponentials by the nonlinear least-squares method with the non-weighted Gauss-Newton algorithm.PET study 2: Within a week of the completion of the PET paradigm study 1, a dopamine PET study was performed in the parkinsonian group. Serial scans  lasting 92 min were performed in the supine position with intermittent arterial blood sampling after a slow bolus injection of 350-MBq of dopamine transporter marker [lsewhere . Briefly3 to k4) of the [11C]CFT in the striatum bilaterally. The value was used as a covariate in further correlation analysis. The reduction of the binding potential was additionally depicted voxelwise using statistical parametric mapping (SPM) software . In this additional image analysis, the side of the reduction contralateral to the more affected limb was set on the right so that the symptom-induced hemispheric side difference was eliminated by flipping the images.Semicircular ROIs were drawn bilaterally over the caudate, putamen, and cerebellum on the MR images as described elsewhere. With theThe CBF data were analyzed using SPM8 without flipping any images. The CBF images were first normalized to the standard stereotaxic Talairach brain atlas and smoothed with an isotropic Gaussian kernel of a full-width at half-maximum of 8 mm. The effects of global CBF were removed by normalizing each voxel count to the total brain count using proportional scaling in SPM. The between-group comparisons  of CBF in each condition were performed by regarding the stabilometer data as confounding covariates using a t-test at each voxel. Clusters of a maximum of 50 contiguous voxels with a p value of <0.05 corrected for multiple comparisons using the FDR method were considered statistically significant.As seen in A comparison of imagining standing with staring in the supine condition (Task2 –Task1) showed a significant rCBF increase in the cerebellar vermis (culmen) in both the PD and NC groups  and diffAs shown in 11C]CFT binding potential in the PD group were significantly lower than those in the normal control group as found in our previous study [11C]CFT binding  was found to be positively correlated with the motor severity of parkinsonism . It is nThere are a couple of limitations. First, the head holder attached to the scanner’s gantry was used to immobilize the subject’s head during standing in the present study, which indicated that standing here was not the same as free standing without support. Currently, though, no immobilization technique during standing exists. Second, no inclusion of patients with postural instability (H&Y 3 or more) cannot provide data for primary involvement of postural disturbance in PD. Third, no measurement during standing with eyes closed did not provide brain activation data under the influence of postural sway for proprioceptive control in balance in PD.In conclusion, we showed abnormal neuronal responses during standing in mildly bradykinetic PD patients in the present study. These early PD patients used their neuronal circuits during motor imaging of standing in a similar manner to healthy subjects. However, standing generated increased activation in the superior part of the posterior cerebellum and the paracentral foot area in the PD patients. This suggested compensatory overactivation in these posture-related areas against the dopaminergic deficiency caused by PD. The hyperactivation in these areas and reduced mesocortical dopamine function reflects a dynamic aspect of posture-related pathophysiology in PD.S1 FigA subject looks at a figure displayed on the monitor (A) and stare at a marker placed 1 meter before the standing point (B). A maximum of 2 minutes was required to stand still.(JPG)Click here for additional data file.


The aim of this study was to assess intra-observer and interobserver variability of biventricular function, volumes and mass in a heterogeneous group of patients with congenital heart disease (CHD) using cardiovascular magnetic resonance (CMR) imaging.CMR imaging provides highly accurate measurements of biventricular volumes and mass and is frequently used in the follow-up of patients with acquired and congenital heart disease. Data on reproducibility are limited in patients with CHD, while measurements should be reproducible, since CMR imaging has a main contribution to decision making and timing of (re)interventions.Thirty-five patients with CHD  were included in this study. A short axis set of contiguous slices was acquired with CMR imaging using a steady-state free precession (SSFP) pulse sequence. Intra-observer and interobserver variability was assessed for left ventricular (LV) and right ventricular (RV) volumes, function and mass by calculating the coefficient of variability.Intra-observer variability was between 2.9% and 6.8%, with the smallest variations in LV and RV end-diastolic volume (EDV) and the highest variations in LV end-systolic volume (ESV) and RV mass. Interobserver variability was between 3.9% and 10.2%, with the smallest variations in LV ejection fraction (EF) and RV EDV and the highest variations in biventricular ESV and mass.Intra-observer and interobserver variability of biventricular parameters assessed by CMR imaging is good for a heterogeneous group of patients with CHD. CMR imaging is an accurate and reliable method for follow-up of biventricular function, volumes and mass and should allow adequate assessment of changes in ventricular size and global ventricular function.Supported by NHF grant 2006B095


Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map2 and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence The online version of this article (doi:10.1038/35057192) contains supplementary material, which is available to authorized users. One important challenge is to define the genetic contribution to human diseases. For many developmental disorders, inherited conditions and cancers, gross chromosomal aberrations provide clues to the locations of the causative molecular defects. These aberrations are visible as alterations in chromosomal banding patterns3 or in the number or relative positions of DNA sequences labelled by fluorescence in situ hybridization (FISH)4. Although tracing gross abnormalities to the level of DNA sequence5 has revealed the genetic causes of many diseases, molecular characterization of chromosomal aberrations has lagged far behind their discovery6. To proceed from cytogenetic observation to gene discovery and mechanistic explanation, scientists will need access to a resource of experimental reagents that effectively integrates the cytogenetic and sequence maps of the human genome.With the draft of the human genome available7). The molecular signatures are STSs (many corresponding to genes or expressed sequence tags (ESTs)), BAC end sequences, or the actual draft or final sequence of the clone  by FISH  and assihe clone . Earlierhttp://www.ncbi.nlm.nih.gov/genome/cyto provides information about all clones in this collection, including how to obtain each clone. -dark and G-light bands of chromosomes  were roughly consistent with at least one FISH observation for 88% of these clones. Collectively, the multisite clones highlight regions that are more likely to become entangled with other regions of the genome during sequence assembly than clones with single FISH locations. Indeed, global BLAST analyses show that regions encompassing sequence tags of multi-site clones (either the sequence of the FISH-mapped clone or a surrogate clone from the assembly) contain blocks of homology found at an average of around 3.9 chromosomal locations . The regions observed by FISH and revealed through homology searches are not fully congruent, however (not shown). These findings indicate that both FISH and sequence analyses may underestimate large-scale duplications and that these complex, inter-related regions of the genome will require special attention during the finishing stages of genome sequencing.The extensive integration of cytogenetic and primary sequence data gives investigators access to fine-structure information—including details on predicted genes—for cytogenetic locations of interest. Tools such as NCBI's MapViewer and the UCSC and ENSEMBL genome browsers see  for URLs18. These clones were mapped with high precision to 850-level bands of varying staining intensity19 on seven chromosomes. The AT content of 58 of the 59 clones in the darkest G-bands exceeds the genome-wide average of 0.59 (mean 0.63), whereas the AT content of only 22 of the 143 clones in G-negative bands is higher than average . These data confirm that dark G-bands are more AT-rich than G-negative bands.This integration provides insight into the sequence differences underlying cytogenetic banding patterns. Sequence analyses of 200-kilobase (kb) regions surrounding the sequence tags of 338 clones mapped with the finest band resolution reveal more striking differences in the base-pair composition between Giemsa-positive and -negative bands than were predicted from earlier studieshttp://dgap.harvard.edu), a breakpoint-spanning clone was identified . The array format offers better sensitivity and resolution22 than metaphase chromosomes, the traditional target for CGH23, and, because the arrayed clones are integrated into the draft, copy-number abnormalities can be related directly to sequence information. To illustrate the power of array CGH, the ML-2 cancer cell line was ‘karyotyped’ using the array. Array CGH revealed relative copy-number losses on 1p, 6q, 11q and 20p and gains of 12, 13 and 20q  . A subset of 442 clones was ordered at very high (∼2–3-Mb) resolution11. FISH analyses were performed using DNA from the bacterial stock used for STS typing. Data that failed to replicate  have been removed. Hybridization to arrays was carried out as described previously29 and by Snijders et al. (in preparation).Details of FISH procedures are provided elsewhereSupplementary Information comprising; Methods, Links, 2 Figures and a Table.


Significance: The reliability of functional near-infrared spectroscopy (fNIRS) measurements is reduced by systemic physiology. Short-channel regression algorithms aim at removing systemic “noise” by subtracting the signal measured at a short source–detector separation  from the one of a long separation . In literature, incongruent approaches on the selection of the optimal regressor signal are reported based on different assumptions on scalp hemodynamics properties.Aim: We investigated the spatial and temporal distribution of scalp hemodynamics over the sensorimotor cortex and evaluated its influence on the effectiveness of short-channel regressions.Approach: We performed hand-grasping and resting-state experiments with five subjects, measuring with 16 optodes over sensorimotor areas, including eight 8-mm channels. We performed detailed correlation analyses of scalp hemodynamics and evaluated 180 hand-grasping and 270 simulated (overlaid on resting-state measurements) trials. Five short-channel regressor combinations were implemented with general linear models. Three were chosen according to literature, and two were proposed based on additional physiological assumptions [considering multiple short channels and their Mayer wave (MW) oscillations].Results: We found heterogeneous hemodynamics in the scalp, coming on top of a global close-to-homogeneous behavior (correlation 0.69 to 0.92). The results further demonstrate that short-channel regression always improves brain activity estimates but that better results are obtained when heterogeneity is assumed. In particular, we highlight that short-channel regression is more effective when combining multiple scalp regressors and when MWs are additionally included.Conclusion: We shed light on the selection of optimal regressor signals for improving the removal of systemic physiological artifacts in fNIRS. We conclude that short-channel regression is most effective when assuming heterogeneous hemodynamics, in particular when combining spatial- and frequency-specific information. A better understanding of scalp hemodynamics and more effective short-channel regression will promote more accurate assessments of functional brain activity in clinical and research settings. Systemic artifacts are typically not constrained locally, but they affect the whole brain and extracerebral tissues, and are thus considered “global.” As a consequence, it is often assumed that global noise is distributed homogeneously over the entire scalp layer and thus that a single global signal can be used for the short-channel regression.In this work, we investigate the scalp hemodynamics above sensorimotor areas with respect to the two differing assumptions of their spatial distribution (heterogeneous versus homogeneous) and their effectiveness for short-channel regression. Through the combination of local and global regressors, we strive to regress systemic signals of cerebral and extracerebral tissue origin more completely. We further hypothesize that the separate consideration of temporal heterogeneities in form of MW oscillations enables a more optimal regression of systemic signals by reducing the physiological noise. To do so, we propose an algorithm based on a general linear model (GLM) that makes use of nonlinear least squares. This work is important as it paves the way for the implementation of more accurate short-channel regression that could increase the overall quality of fNIRS measurements, allowing more wide-spread application of this technology. This can particularly help to promote clinical applications by making measurements more reliable at the individual level.2To investigate scalp hemodynamics over sensorimotor areas and its influence on the estimation of the functional HR, we conducted an fNIRS experiment to obtain resting-state and motor-execution measurements. The study was conducted in accordance with the Declaration of Helsinki Ethical Principles and Good Clinical Practices, and all subjects provided informed consent.2.1Five male, right-handed volunteers . The system measured with eight sources and eight detectors at two wavelengths (760 and 850 nm), with a sampling frequency of 7.8125 Hz. For the conduction of this study, an fNIRS cap enabling the measurement with SS and LS channels was deployed. The cap was provided by the manufacturer and enabled the concomitant measurement with 20 LS channels with a source–detector separation of 30 mm and 8 SS channels of 8-mm distance that were located around each light source [lacement . The oth2.2Data were processed using custom-built MATLAB scripts . Channels with low signal quality were excluded from data analysis. They were identified based on the evaluation of the cardiac signal by the method of Perdue et al.,,,,,Motion artifacts were minimized with a movement artifact reduction algorithm involving detection by moving standard deviation and removal by spline fitting.The LS channel with the strongest cerebral activation during the hand-grasping experiment according to GLM see Sec.  was sele2.3,,–The GLM method is a commonly known technique that allows the regression of short channels and that is relatively easy to implement.2.3.1In GLM, the measurements (To solve Eq. (1) and obtain an estimated parameter An estimated, cerebral activity Three approaches based on literature used the standard GLM method: (i) selecting a local regressor (2.3.2The two other GLM methods were based on the assumption of phase-shifted oscillations in the MW band.–Non-negative least squaresTo prevent a biased estimate by restricting The fourth (iv) GLM approach (method: The fifth (v) approach  convolved with the HR function was superimposed on the measurements from the resting-state run.,,We deliberately used real resting-state measurements instead of purely simulated systemic signals, because we expected that simulated noise could only insufficiently reflect heterogeneous behavior in the scalp and brain layers. A possible limitation of this approach is that resting-state measurements may contain spontaneous neural activity with amplitudes comparable to functional brain activity,2.5The normalized MW amplitude Pearson’s correlation coefficient  and (3) was used, with the design matrix To fathom the potential of the regression approaches for protocols that are based on single-trial evaluation, such as may be the case for brain–computer interfaces (BCI), we additionally calculated a trial-based contrast-to-noise ratio (CNR) metric. The results of the CNR evaluation are presented in the 3The results section is split into three parts. First, the spatiotemporal distribution of scalp hemodynamics over sensorimotor areas is presented with respect to the incongruent assumptions of heterogeneity and homogeneity Sec. . Second,3.1The normalized MW amplitudes  were investigated for the same frequency bands  and experiments  as before, and are shown in 3.2In this section, we report on the effectiveness of five GLM regression approaches to remove physiological artifacts from simulated measurements. For each subject, a total of 54 HRs were simulated, which were overlaid on resting-state measurements of a reference LS channel over the left primary motor cortex. All regression approaches were applied on the same time courses, but used different sets of SS signals . For each subject, a All GLM regression approaches enabled the more effective estimation of brain estimates in comparison to the original LS signal , expressed as significant differences for all metrics in The additional inclusion of MW oscillations in 3.3The block averages of relative concentration changes for the five subjects in All GLM methods achieved better estimates of brain activity than the unregressed original LS signal in ons Sec. , and maiThe additional inclusion of MW oscillations in 4A better understanding of systemic hemodynamics is crucial for the correct interpretation of fNIRS measurements and, thus, the future usability of fNIRS for research and clinical applications. While short-channel regression has been identified as an important step in making fNIRS technology more applicable, the important question of which systemic signals to regress remains insufficiently addressed. In this work, we investigated in detail the fNIRS signals from 8 SS channels (8-mm source–detector separation) over sensorimotor brain areas by comparing them with a reference LS signal  during resting-state and a hand-grasping experiment. We evaluated five GLM regression approaches that made use of different regressor signals, which were selected from literature and physiological assumptions. We proposed two regression approaches based on non-negative least squares to include additional spatial and temporal information of the scalp  compared to state-of-the-art approaches. We show the improved effectiveness to remove physiological noise, thereby shedding light on the optimal selection of scalp regressors to obtain better estimates of functional brain activation.4.1,,,–,,,,With respect to the incongruent assumptions on the spatial distribution of scalp hemodynamics  requiring “clean” signals to minimize false positives and false negatives.4.4This study is limited due to the relatively low number of participants. Nevertheless, a comprehensive view of the generalizability of our results is obtained. The subjects had very different signal contents: subjects 1 and 2 had strong HRs, subjects 3 and 5 had large MW amplitudes, and subject 5 had very strong task-evoked systemic activation. Nevertheless, the two ,The MW-bandpass filtered signal may not solely include MW oscillations, but also contributions from task-evoked systemic activation and the HR (contained in the LS channel). Therefore, there is a risk that removing specific frequencies may alter the magnitude or slope of the HRs after regression.,,,In the simulation part of this work, we did not assume any systemic task-evoked activation, which minimizes the possibility to overcompensate systemic signals. It is known that during a functional task, the autonomic nervous system activity is increased,,,,In this study, we deliberately used a simple regression method and performed the regression offline on the entire dataset. Future studies should compare our findings with other approaches proposed in the literature, such as autoregressive models,,,The hardware employed provided SS channels next to the sources but not next to the detectors. Results may have looked different with additional SS channels located at the detectors.5With this work, we aimed to shed light on the behavior of scalp hemodynamics over the sensorimotor cortex and the influence of scalp regressors on short-channel regression. The better understanding of the systemic physiology enhances the estimates of functional cerebral activation, and is an important step in promoting routine application of fNIRS, especially at the individual level as required in clinical applications. We conclude that1.The scalp hemodynamics follows a heterogeneous and frequency-specific behavior. These heterogeneities are superimposed on a global, close-to-homogeneous distribution.2.The introduction of an adapted GLM approach using non-negative least squares enabled the inclusion of multiple regressors with a reduced risk of overfitting compared to state-of-the-art methods. We tested five regressor combinations and found that better performance was achieved when assuming heterogeneous scalp hemodynamics. In particular, we showed the benefit of considering delayed MWs in the regression to compensate for their phase-shifted oscillations between different scalp regions and compartments .With this work, we highlighted the importance of applying short-channel regression and present a way to unite the multilayered behavior of systemic signals in a regression algorithm. We proposed an easy-to-implement short-channel regression method based on GLM and showed the benefit of including multichannel and multifrequency information. We encourage all future fNIRS studies to concomitantly capture SS channels to reduce the influence of systemic physiology.6A CNR metric was calculated to investigate the effectiveness in improving single-trial estimation of brain activity. A high CNR value gives an indication on the ability of single-trial classification, for example in the frame of a BCI.,,,The trial-based CNR metric was established in reference to Saager et al.,Results in In


Recently, an increased interest toward enzymatic carboligation was observed, as biocatalytic carbon–carbon bond formation is a common obstacle in retrosynthetic planning. The construction of extended 2-oxoacid frameworks by 2-oxoacid aldolases and enzymes acting as aldolases is a potent tool for synthetic chemists since a broad spectrum of downstream reactions through functional group interconversions gives access to a plethora of compound classes. In the search for selective biocatalysts, successful protein engineering efforts and high throughput screenings from biodiversity expand the structural diversity of nucleophile and electrophile substrates. Several successful examples with an emphasis on reactions catalyzed by class II aldolases and enzymes acting as class II aldolases are highlighted, including reactions in which both enantiomeric products and in selected cases even diastereomeric products are accessed. As biocatalysis is progressively implemented in pharmaceutical processes it is notable that many functional group interconversions are feasible; however, the demand in selective carbon—carbon forming reactions remains underexploited –5. The bd-glucarate aldolases (GarL) in the catabolism of d-glucarate/galactarate  mechanistic type adopt the common TIM barrel fold and are hexamers  binding one metal cofactor per subunit. The metal itself binds by an aspartate and a glutamate over a water bridge. An arginine residue in the active site is responsible for the binding of the nucleophile carboxylate and together with an adjacent histidine are identified to be crucial for enzymatic activity, participating in the activation of metal-bound water for proton transfer during catalysis ,20. HencS-configuration and the R-configuration at the 2-hydroxy group in the electrophilic aldehyde. Furthermore, although both syn- and anti-diastereoselectivities are described, the configuration of the chiral center at C3 is exclusively S-oriented . A . A 29]. leophile . Furtherde may be reduced over time toward the thermodynamic product and therefore requires kinetic control . In a co-factor independent mechanism KphMTE.coli utilizes a Lewis-acidic divalent transition metal such as cobalt to act as a class II aldolase . Co. CoS.wit2O2 allows the synthesis of a carboxylic acid with a loss of a carbon atom and enables subsequent esterification, which reduces the polarity of products [ee in the product [The oxidative decarboxylation with stoichiometric concentrations of Hproducts ,35,38. SThe implementation of inherently sustainable enzyme-driven catalysts into chemical processes is advancing with the recent success in the development of novel biocatalytic carbon—carbon bond-forming reactions ,11. Recein situ would boost the applicability of pyruvic acid aldolase reactions [Biocatalytic aldol reaction still has limitations but is clearly on the upswing. One challenge is the supply of highly oxidized starting material for aldol reactions. Therefore, upstream reactions which provide the tedious 2-oxo acid and aldehyde substrates eactions . It also


Accurate diagnosis at an early stage of infection is essential for the successful management of any contagious disease. The coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is a pandemic that has affected 214 countries affecting more than 37.4 million people causing 1.07 million deaths as of the second week of October 2020. The primary diagnosis of the infection is done either by the molecular technique of RT-qPCR by detecting portions of the RNA of the viral genome or through immunodiagnostic tests by detecting the viral proteins or the antibodies produced by the host. As the demand for the test increased rapidly many naive manufacturers entered the market with novel kits and more and more laboratories also entered the diagnostic arena making the test result more error-prone. There are serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on control strategies. The significance of the test is also complexed by the presence of asymptomatic carriers, re-occurrence of infection in cured patients as well as by the varied incubation periods of the infection and shifting of the viral location in the host tissues. In this paper, we review the techniques available for SARS-CoV-2 diagnosis and probable factors that can reduce the sensitivity and specificity of the different test methods currently in vogue. We also provide a checklist of factors to be considered to avoid fallacious practices to reduce false positive and false negative results by the clinical laboratories. Nidovirales and family Coronoviridae and subfamily coronavirinae that includes four genera. The SARS-CoV-2 belongs to the genus Betacoronavirus and subgenus Sarbecoviruses and is related to other human coronaviruses like SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and carries an enveloped, non-segmented positive strand RNA genome of 30 kb. The viral genome codes for four structural proteins and sixteen nonstructural proteins [The coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2 virus) is believed to have originated in Dec 2019 at Wuhan city, China [proteins .To enable management of a pandemic outbreak, rapid and accurate diagnosis is of paramount importance. Initial clinical/symptomatic investigations depended on blood count, coagulation profile, serum biochemical tests like Liver Function Test (LFT) and Renal Function Test (RFT), creatine kinase, lactate dehydrogenase, and other electrolytes. Respiratory specimens including nasal and pharyngeal swabs, bronchoalveolar lavage fluid, sputum, or bronchial aspirates were tested for common viruses, including influenza, avian influenza, respiratory syncytial virus, adenovirus, parainfluenza virus, SARS-CoV and MERS-CoV using Reverse Transcription quantitative PCR (RT-qPCR) assays approved by the China Food and Drug Administration. Routine bacterial and fungal examinations were also performed.The World Health Organization (WHO) declared COVID-19 as a pandemic on 11 March 2020 . Even thN), the spike protein gene (S), the envelope protein gene (E), RNA dependent RNA polymerase (RdRp) and part of the major ORF1a/b. The majority of the countries that are practicing population quarantine are developing strategies to come to normalcy for which appropriate sensitive and specific diagnostic tests are essential.The etiological agent of COVID-19 is diagnosed in the clinical laboratory either through the presence of selected genes of the viral genome, selected viral proteins or by the antibodies produced by the infected host. The major genes targeted for the detection of the viral genome are the nucleocapsid protein gene  targeting different gene/genes. Majority of the tests use an ry tests .Reverse Transcriptase quantitative PCR (RT-qPCR) for SARS-CoV-2 diagnosis: Reverse Transcriptase quantitative PCR (RT-qPCR) is the first choice and globally accepted diagnostic assay for the screening of the SARS-CoV-2 virus. The pandemic nature of the SARS-CoV-2 outbreak of 2020 has sparked RT-qPCR testing of the virus on an unprecedented scale globally. Kary Mullis, the originator of the Polymerase Chain Reaction (PCR) might not have thought even in his wildest dreams that the technique he invented will become the most widely used diagnostic test by mankind. The unforeseen and urgent demand for SARS-CoV-2 testing has stretched diagnostic RT-qPCR resources and personnel to the limit and warranted quick deployment of new testing facilities and personnel to the scene. The major RT-qPCR assays available for SARS-CoV-2 detection are listed in The success of the diagnostic procedure depends on the collection of appropriate specimens from the right anatomic location/organ at the right time of infection. The initial studies on the bio-distribution of SARS-CoV-2 in various patient samples during the outbreak in the Hubei, Shangdong and Beijing in China revealed the highest PCR positive rates in bronchoalveolar lavage (BAL) 93%), followed by sputum (72%), nasal swabs (63%), pharyngeal swabs (32%) and feces (29%) . Another3%, folloRespiratory sampling is the most common and widely accepted method for SARS-CoV-2 detection by RT-qPCR and can be broadly divided into upper respiratory and lower respiratory sampling . Upper rNS, NPS and OPS are taken with flocked swabs. The upper respiratory sampling is comparatively easier than the lower respiratory sampling but need trained personnel. The discomfort created for the patients by the procedure could induce sneezing or coughing, which can lead to aerosol exposure of the virus . It has BAL is reported to have a higher diagnostic value than upper respiratory samples in patients with pneumonia . HoweverThe widely adapted NPS or OPS swabs testing for SARS-CoV-2 needs interaction of health care workers with patients and requires specialized swabs and personal protective equipment, which are in short supply. This has led to exploration of other types of samples that patients can collect on their own, without discomfort and minimizing the risk of nosocomial infections. Of late, deep throat saliva (DTS) was also reported as an alternative for patient self-collected specimens with less health hazard to workers and without compromising diagnostic results . HoweverOral fluid and saliva have also recently been proposed as potential samples that the patients could collect by themselves and had shown similar diagnostic sensitivity to NPS ,17. For Although self-collected patient samples have advantages like wider reach especially for the elder patients, for risk mitigation of exposure to the virus, the technical quality of specimen collection has to be considered especially for nasopharyngeal and oropharyngeal swabs. In a study in Iran of 50 adult patients, it was revealed that there was discordance in the result between professional-collected and patient-collected samples. Many oropharyngeal and nasopharyngeal swabs that were negative in patient-collected samples were positive when collected by laboratory professionals . In the 5 copies per whole swab with maximum load detected on the fourth day being 7.11 × 108. After day five the load of samples decreased with an average viral load of 3.44 × 105 copies per swab and a detection rate of 39.93%. The last sample that was tested positive was taken 28 days after onset of symptoms. The study also revealed that there was no significant difference in viral load or detection rates between nasopharyngeal and oropharyngeal swab samples. When compared to the SARS virus, peak viral loads were detected much earlier for SARS-CoV-2 (before day 5 for SARS-CoV-2 and days 7–10 for SARS). The concentration of virus was also much higher for SARS-CoV-2 (1000 times more than SARS). In the German study, sputum samples had an average viral load of 7 × 106 copies/mL and a maximum of 2.35 × 109 copies/mL and the decline in viral load was slow compared to swabs. In two-thirds of the patients (6/9), sputum and stool remained positive for viral RNA by PCR for more than three weeks [Virological assessment of individual SARS-CoV-2 positive samples by two independent laboratories in Germany provided insights into the viral load in patients . Swabs wIn a study of 213 patients in China, sputum samples provided a higher rate of accuracy in positive diagnosis (75 to 90%), when compared to nasal swabs (53 to 73%) during the first 14 days after the onset of symptoms . HoweverProper sampling techniques using appropriate swabs are essential for high-quality upper respiratory specimens, otherwise it will lead to false RT-qPCR negatives. CDC recommends only synthetic fiber swabs with plastic or wire shafts and strongly advises against using calcium alginate swabs or swabs with wooden shafts as they can inactivate some viruses and inhibit PCR reactions . The swab should be inserted through the nostrils parallel to the palate until resistance is encountered. Swabs should reach a depth equal to distance from the nostrils to outer opening of the ear. After collection, the swabs must be placed in the viral transport media (VTM) or Universal Transport Media (UTM), and stored and transported as per the guidelines recommended by CDC/WHO. For swabs in VTM, storage at 2–8 °C is recommended for up to 72 h and −70 °C for longer duration. Considering the fact that many of the mass samplings for SARS-CoV-2 is done at outdoor/mobile/makeshift sampling stations outside proper clinical facilities, care should be taken to store and transport the swabs at the recommended temperatures to the testing facilities where RNA isolation is carried out to prevent false negatives due to sample degradation. Appropriate biosafety protocols for the COVID-19 specimen samples from collection, transport and processing have been suggested to prevent laboratory-associated SARS-CoV-2 infection .Betacoronavirus, the proteinase K and DNase I method of homogenizing sputum reduced false negatives in spiked sputum samples [Isolation of RNA is the initial step of any quantitative reverse transcriptase PCR assay. This is one of the most critical steps determining the reproducibility and biological relevance of the results. RNA, unlike DNA, is highly susceptible to degradation. So the sample storage, handling and RNA isolation steps have to be carried out with optimized protocols minimizing the loss of RNA due to degradation at each step. The isolated RNA must be of high quality, without any nucleases or substances that may inhibit cDNA synthesis. Among clinical samples, mammalian blood, in particular, the heme compound is a well-known PCR inhibitor. RNA isolation from respiratory samples like sputum is reported to have yielded false-negative results due to challenges in isolating RNA from mucus containing sputum . Additio samples . Hence, The pandemic nature of the SARS-CoV-2 outbreak has resulted in an unprecedented demand for viral RNA based diagnostics and has resulted in a shortage of RNA isolation kits/reagents. In such a scenario, various research groups have come up with direct RT-qPCR assays skipping the RNA isolation process. Some of these tests have accurately identified 92% clinically known positive samples without the need for RNA isolation ,26. In cP mRNA is used in RT-qPCR as an internal house keeping control. Positive RNA controls can be spiked samples with synthetic SARS-CoV-2 RNA or previously validated positive samples. The demand for RNA isolation from a huge number of samples resulted in the adaptation of a wide variety of RNA isolation systems—automated and manual and some of which may lead to aerosol contamination of samples due to improper use. A previously validated negative patient sample serves as negative extraction control for RNA isolation to rule out such issues [Positive and negative controls in RNA isolation are of paramount importance due to the huge numbers of samples tested, wide variety of specimen types, and different types of RNA isolation systems. To ensure sufficient human RNA is obtained from the specimen, RNAse h issues . A no-teFor the detection of RNA viruses like SARS-CoV-2 reverse transcription qPCR is recommended as the most sensitive method. The isolated total RNA  is reverse transcribed into a cDNA first using reverse transcriptase enzyme. PCR or qPCR is performed using the cDNA as a template to exponentially amplify the target gene of interest. In the beginning, this was a two-step process with the cDNA synthesis performed separately first using the isolated RNA, and aliquots of the cDNA subsequently used for PCR or qPCR. Reverse transcriptase of viral origin from Avian Myeloblastosis Virus (AMV-RT) and Moloney Murine Leukemia Virus (MMLV-RT) were the two enzymes widely used. AMV-RT has a higher reaction temperature, thereby reducing problems associated with RNA secondary structure. MMLV-RT has lower RNAse H activity than AMV-RT and hence has better yield and longer size of cDNA products . Newer gReverse transcription can be carried out by using different primers—oligo-dT, random primers or gene-specific. The choice of the primer depends on the type of RNA, cDNA yield, and specificity. Use of oligo-dT primer for RT will make the cDNA synthesis for mRNA and not for ribosomal RNA or viral transcripts or RNA transcripts with significant secondary structure. Random primers bind at multiple portions of the RNA transcript and are not specific. However, they yield the most amount of cDNA and can bind with a wide variety of RNA like rRNA, viral RNA and RNA with secondary structure . The useRNase P gene control serves as an important sample extraction quality control. Along with the viral RNA, the patient’s RNAse P mRNA is also isolated during the RNA isolation step. The RNAse P mRNA is reverse transcribed into cDNA and the RNAse P Primers amplify a fragment of this gene, which is then detected by real-time PCR. As per the CDC guidelines, all clinical samples tested should have Ct values less than 40 for RNase P in RT-qPCR. Failure to detect RNase P gene within the prescribed limits may indicate improper RNA extraction, RNA degradation/loss, absence of sufficient human cellular material due to improper collection, reagent or equipment malfunction. It may be noted that the RNase P gene control PCRs are run in parallel with the SARS-CoV-2 and not multiplexed in the same PCR reaction along with the SARS-CoV-2 target genes as per CDC/WHO recommendations. The relative high abundance of human RNase P RNA in the sample when compared to SARS-CoV-2 viral RNA may cause reduced sensitivity of SARS-CoV-2 target genes when multiplexed.The use of In this unprecedented testing on a global scale for SARS-CoV-2, it is critical to maintain proper quality control standards in RT-qPCR. The assays are mostly performed in multi-well plates and are prone to aerosol contamination. RT-PCR negative control is usually nuclease-free water and a proper negative control should not give any amplification signals. In-vitro synthesized RNA transcripts based on the SARS-CoV-2 are used as positive control samples and are expected to give Ct (threshold cycle) in the recommended ranges by different assay kits . Any ampnucleocapsid gene (N1 and N2). A third primer-probe set for the human RNase P gene serves as a control for RNA extraction and cDNA synthesis. The WHO recommends the use of a different set of SARS-CoV-2 genes; RNA-dependent RNA polymerase (RdRP) and envelope (E) genes [For RT-qPCR, CDC recommends primer-probe sets for two different regions of the SARS-CoV-2 E) genes . These rE) genes . CDC andE gene primer-probe set [N gene, is also reported by SARS-CoV-2 testing centers in Korea, who suggest that the N gene is 7–43 fold more sensitive [A recent comparative study from Yale School of Medicine with different primer/probes for SARS-CoV-2 detection revealed that sensitive primer-probe sets were E-Sarbeco, HKU-ORF1, and 2019-nCoV_N1. The RdRp-SARS had the lowest sensitivity that could be attributed to a mismatch in the reverse primer . In anotrobe set  and the robe set . The insensitive .Taq DNA polymerases, and thermal cyclers used. The Charite paper suggests the following PCR cycling conditions: reverse transcription at 55 °C for 10 min and initial denaturation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 15 s, 58 °C for 30 s [Taq polymerases with different reaction conditions. These kits are validated at specific conditions recommended by the manufacturers based on the assay design/reagents used. Hence, users should follow instructions and reaction conditions recommended for the individual kits/reagents they are using and should not follow a general protocol for PCR amplification.Reaction conditions for COVID-19 RT-qPCR depend on the target gene, primers, reverse transcriptases, for 30 s . CDC recfor 30 s . It mustEven before starting the RT-qPCR run, the data capture settings in the qPCR software should be accurately set to capture the data in real-time. CDC provides clear guidelines on instrument setup, entering fluorescent detector, sample setup and reaction conditions in the real time PCR software . The tarThe Ct (threshold cycle) value is the most commonly used parameter for reporting diagnostic qPCR results. Ct is the cycle when the sample fluorescence exceeds the background fluorescence by a chosen threshold . Most reCDC provides clear guidelines on the interpretation of assays and results with threshold and Ct values. The No Template Control (NTC) without the RNA for all the primer and probe combinations should not have fluorescence amplification curves that cross the threshold line. If any NTC reactions show fluorescence that crosses the threshold level, contamination needs to be suspected and assays repeated. The SARS-CoV-2 positive control samples should have a Ct value of less than 40, well above any background fluorescence. Reagent failure or primer or probe integrity needs to be suspected in case of any Ct value beyond 40 or lack of well-defined amplification curves for the positive controls. The human RNAse P (RP) controls are also expected to show amplification curves that cross the threshold within the 40 cycles. Failure to detect RNAse P could be due to poor collection method yielding insufficient cellular material, improper nucleic acid extraction from the specimen and instrument/reagent/assay malfunction. Ideally in an assay, SARS-CoV-2 positive control samples and RNAse P control should have Ct values <40 and NTC controls should not have fluorescence amplification curves that cross the threshold line within 40 cycles. A clinical specimen is considered clear positive when all the SARS-CoV-2 target genes and RNAse P have Ct value < 40. Additional scenarios with Ct values and interpreting them are discussed in detail in the CDC guidelines .Apart from the RT-qPCR for SARS-CoV-2, which is considered the gold standard and most widely used, there are many other nucleic acid detection technologies for the virus that are summarized below.Loop-mediated Isothermal Amplification (LAMP) is a cost-efficient one-tube isothermal nucleic acid amplification technique carried out at a single temperature that amplifies DNA fragments of interest with high specificity and sensitivity. In the original method, four different primers, recognizing six distinct regions of the DNA were used along with a DNA polymerase with high strand displacement activity . The LAMAlthough RT-qPCR is currently the gold-standard in SAR-CoV-2 diagnosis, the protocols are time-consuming, laborious and need specialized expensive equipment and trained personnel. Well validated RT-LAMP assays for SARS-CoV-2 would be simpler, cost-efficient, speedy and equally sensitive alternatives to RT-qPCR. For the detection of RNA viruses like MERS Coronavirus, reverse transcription has been successfully coupled with LAMP to create a one-pot RT-LAMP assay that had been validated  much befORF1ab and S genes. The assay did not have cross-reactivity to 60 other respiratory pathogens and was validated with 130 COVID-19 positive clinical RNA samples already tested by qPCR. The assay showed 100% sensitivity and specificity and gave results within 30 min [(N) protein gene with a detection limit comparable to RT-qPCR. The assay was evaluated on 156 clinical samples with sensitivity of 100% and specificity of 98.7% [Using the sequences from 103 SARS-CoV-2 genomes, a LAMP assay was developed by a group in China using primers specific to n 30 min . Researcof 98.7% . Anotherof 98.7% .ORF1a, N, and E genes) and a human actin control. Multiple samples (up to 96) are amplified by multiplexed barcoded loop-mediated amplification and SARS-CoV-2 detected by real-time nanopore sequencing in less than two hours on a single MinION flow cell [Recently LAMP has been coupled with nanopore sequencing to develop LAMPore Assay for detection of the SARS-CoV-2 virus. The LAMPore SARS-CoV-2 is a multiplex assay that targets three different regions of the virus genome -Cas (CRISPR-associated) systems in prokaryotes have evolved as an adaptive immune system against invading nucleic acids and this technology has been widely adapted for genome engineering in most living organisms . DiffereN gene of SARS-CoV-2 in three simple steps at a sensitivity of 100 viral genomes per reaction and does not need separate viral extraction with specialized equipment. The virus is lysed quickly to release the RNA from the patient sample, followed by RNA detection using STOP Covid and finally visualization of the result using a lateral flow dipstick in 70 min [SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology combines isothermal pre amplification with Cas13 to detect single molecules of RNA or DNA at attomolar levels and has been adapted to detect infectious viruses like Dengue and Zika. A field-deployable multiplex viral diagnostic platform was developed by integrating SHERLOCK with HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) protocol for viral diagnosis from bodily fluids with minimum equipment and sample processing . A STOP n 70 min . This kiN and E genes was developed and validated by Mammoth Biosciences and the University of California [Another parallel developed method, DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), uses Cas12a ssDNase activation with isothermal amplification for rapid and specific detection of human viral pathogens with attomolar sensitivity . A SARS-lifornia . This raFrancisella novicida (FnCas9) that has been reported to have higher target specificity and negligible affinity with off-targets, when compared to widely used Streptococcus pyogenes Cas9 systems [NSP8 regions of SARS-CoV-2 for rapid diagnosis [This method uses Cas 9 systems from the bacterium  systems . Utiliziiagnosis .A microfluidic system for massive multiplexed simultaneous detection of pathogens was developed by the Broad Institute of MIT and Harvard utilizing the specificity of the SHERLOCK Cas13. The CARMEN  platform uses microwell array chips having 177,840 microwells . In thisTrueNat is a recent test developed by Indian scientists by adapting the test already in vogue for pulmonary tuberculosis to COVID-19. TrueNat is a chip-based portable PCR test developed for quick and affordable molecular pathogen detection for use in low infrastructure health facilities in developing countries. BigTec Laboratories, Bangalore, India, developed this technology and earlier adapted this in 2014 for early Tuberculosis detection . The sysThe World Health Organization recommends aggressive testing as one of the main strategies against the COVID-19 pandemic. The overwhelming demand for mass screening by RT-PCR testing for the virus made the global healthcare system struggle with a shortage of testing kits and trained personnel . To expaOne of the first pool PCR testing for SARS-CoV-2 was done in Israel in March 2020. Researchers pooled individually isolated RNA samples that were earlier confirmed as SARS-CoV-2 positive or negative and tested the pools by RT-qPCR. They found that a single clinical SARS-CoV-2 RNA sample can be consistently detected in a pool of up to 32 samples with an estimated false-negative rate of 10% . In the In-silico analysis, using an application called Shiny, estimated the optimum pool size to be five specimens to detect at an assay sensitivity of 95% or 100% and 100% specificity, when the virus prevalence rate is set at 5%. Twenty-five experimental pools created with one known positive specimen and four negative specimens were positive with pooled Ct values with an average difference of 2.45 of the individual specimen Ct values and range of difference from 0 to 5.03. Using this five specimen pool strategy they tested 60 samples using only 22 extractions/PCR tests and identified two positive individuals. They estimated that when the rate of infection of SARS-CoV-2 is less than 10% in the population, the five specimen pool strategy can increase the testing efficiency by 133% and reduce the number of tests by 57% [s by 57% .Mass testing by real-time qPCR for SARS-CoV-2 is laborious, expensive, and has led to a global shortage of diagnostic reagents. With the number of infections on the rise, and a large number of asymptomatic people also to be tested, pool testing will provide a strategy to extend the testing with the existing resources. In countries like India, with a huge number of people, pool testing is adopted by some states and the Indian Council of Medical Research (ICMR) has provided an advisory on using pooled samples . As per the ICMR guidelines, pool PCR testing is recommended for use in areas of low positivity (<2%) and pooling size has to be kept to a maximum of five samples to avoid over dilution leading to false negatives.Although pool PCR is an interesting strategy to increase the testing scope and improve efficiency with limited resources, it has to be carried out with utmost care by competent testing facilities, without compromising the sensitivity and accuracy.The primary transmission of the SARS-CoV-2 virus is believed to be through direct contact and respiratory droplets. However, aerosol transmission and indirect-contact based transmission is also presumed to be involved in the rapid spread of the disease. In a study in Israel, viral RNA could be detected in 52.7% surface samples from surroundings of symptomatic COVID-19 patients in isolation units and 38% from surfaces in quarantine hotels of asymptomatic and mild COVID-19 patients . PotentiIn the midst of the COVID-19 outbreak, clamor for serological tests also increased either for antibody or antigen detection . SerologELISA kits for SARS-CoV-2 antibody detection made by different vendors have been certified by various regulatory agencies. However, for the detection of SARS-CoV-2 antigen only limited ELISA kits are available in the market. ELISA based serological tests can be employed to detect viral antigen in the clinical samples, such as swab, lung lavage, and sputum, or it can be employed to detect the presence of antibodies in the plasma or serum. These tests currently available differ in many respects such as the antigen used, detection method used and the solid matrix employed for capturing antigen/antibody, etc. Apart from well-established mature technologies, novel methods, such as microfluidic chips, plasmonic fiber-optic absorbance biosensor, and field-effect transistor (FET)-based biosensing devices, are being investigated by different laboratories ,61,62,63Several firms have produced ELISA tests to detect SARS-CoV-2 antibodies based on the classical indirect ELISA method. In indirect ELISA the antigen is coated on ELISA plates to capture virus-specific antibodies, which in turn are detected by secondary conjugated antibodies. Most of the kits available use recombinant spike protein or nucleocapsid protein as the capture antigen. Based on the antigen used to capture, the sensitivity and specificity of the ELISA can vary widely . Some ELThese are mostly used for the detection of specific subtypes of antibodies such as IgM, IgG or IgA in the serum. Polystyrene microwell strips are pre-coated with antibodies directed against the specific subtype  of human immunoglobulin proteins. During the first incubation after addition of specimen (patient’s serum/plasma), specific subtype antibodies will be captured inside the wells. After washing out all the other substances of the specimen and other antibody subtypes, the antibody subtype-specific to SARS-CoV-2 captured on the solid phase is detected by the addition of SARS-CoV-2 antigen conjugated to the enzyme or other reporter assays .Third generation ELISA is known for its higher sensitivity and specificity . Here, pThe classical ELISA employs plastic micro well plates with polystyrene surface to immobilize the antigen. Majority of ELISA kits available in the market are of this kind. This ELISA format was there in the diagnostic field for a long time therefore the procedure has become standardized and most labs are well equipped to carry out the task in terms of equipment and training. However, these are not easily amenable to automated high throughput systems that process hundreds of samples a day, which is the urgent need in the face of the pandemic. Novel formats use magnetic beads or polystyrene beads as a solid matrix to immobilize antigen or antibody depending on the assay. Apart from high throughput, this method allows multiplexing the assays thereby reducing sample volume required. Shorter turnaround time is the main advantage of these automated systems .ELISAs in the market differ in the methods used for detection. The detection methods of ELISA have undergone huge improvements from the traditional enzyme-mediated colorimetric method. Novel automated systems use a more sensitive chemiluminescence detection method, which offers 100 fold more sensitivity and allows automation. Chemiluminescence systems have a detection limit of up to 170pg/mL of the analyte. Abbott chemiluminescence immunoassay is the only system approved by the EU for the detection of COVID-19 antibodies . MAGLUMIFluorescent labels instead of enzymes allow for multiplexed ELISAs, which enable estimation of antibodies against more than one antigen in a single assay or more than one antigen in the matrix. Conjugation with fluorophores with different spectral properties enables quantification of different analytes in a single reaction thus enabling gain on the information in limited turnaround time. The fluorescent immunoassay uses SARS-CoV-2 Ag, covalently linked to polystyrene microspheres. The microspheres are then mixed with serum to bind SARS-CoV-2 antibodies present. Non-bound antibodies are washed away and then bound antibodies are allowed to react with a biotinylated secondary anti-human immunoglobulin reagent . After washing, microspheres are mixed with phycoerythrin labeled streptavidin and bound antibody is detected with the Luminex cytometer, or other instruments. The fluorescence measured is proportional to the amount of anti-SARS-CoV-2 antibodies in the serum .® immunoassays are designed for the detection and quantification of target molecules in biological samples. These chemiluminescence no-wash assays are ideally suited for miniaturization and automation. It relies on amplified luminescent proximity homogeneous assay. In a sandwich AlphaLISA assay the target is captured by a biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads. The binding of the two antibodies to the analyte brings donor and acceptor beads into proximity. Laser irradiation of donor beads at 680 nm generates singlet oxygen, triggering a cascade of chemical events in nearby acceptor beads, which results in a chemiluminescence emission at 615 nm. In competitive AlphaLISA immunoassays, a biotinylated analyte bound to streptavidin donor beads is used with an antibody conjugated to AlphaLISA acceptor beads. AlphaLISA has higher sensitivity and shorter turnaround time as it removes washing steps [AlphaLISAng steps . HoweverThe SARS-CoV-2 virus codes for around 20 proteins including 16 nonstructural and 4 structural proteins. The body will potentially produce antibodies against all these proteins and the serum will contain antibodies against all these viral proteins. However, these differ in their abundance and suitability to differentiate from SARS-CoV-2 from other coronaviruses infecting humans. Most of the kits use spike protein (S), or nucleoprotein (N) as antigen. The nature of antigen used has an important bearing on the sensitivity and specificity of the ELISA. The antigen chosen should be able to detect and differentiate from closely related viruses, in this case, common coronaviruses  or SARS-CoV (HKU-1) . It has The lateral flow tests or immunochromatographic tests sometimes called lab on a chip, contains all reagents required to carry out the test in a simple plastic cassette. The principle behind LFA is that a liquid sample containing the analyte of interest moves by capillary action through various zones of the polymeric membrane, on which the molecules that can bind with the analyte are attached or impregnated at a specific location. One of the major advantages of this technique is that no elaborate sample processing step is needed, which is the main drawback of other tests such as ELISA or RT-qPCR. Other advantages of lateral flow testing kits are low cost, simple single-step procedure, requirement of small sample volume, ability to be deployed at the point of care, uniformity of sample used, etc. An added advantage of LFT is the long shelf life of the cassette without refrigeration .Immunochromatographic tests for antigen are simple tests, where a nitrocellulose test strip is impregnated with a reporter  conjugated to the antibody. In the rapid CoV-2 test, anti-SARS-CoV-2 specific monoclonal antibody either against viral spike protein or nucleocapsid protein is used to impregnate the nitrocellulose filter. The test strip is then dipped in the clinical sample  and mixed with a buffer. As the liquid moves through the membrane by capillary action, the viral antigen present in the sample reacts with the antibody and forms a complex. The complex is then captured by the second antigen-specific antibody immobilized at the detection line. LFA works on a similar principle of an immunochromatographic test but the sample suspended in the buffer is added by dropper to the sample pad.The detail of sampling for antigen testing is similar to the one used for RT-qPCR and has been mentioned under the Molecular Testing. Sampling is a critical part of any diagnostic assay. For SARS-CoV-2, the WHO recommends a nasopharyngeal swab as the ideal sample. However, throat swabs, sputum, and tracheal aspirates are also being used as clinical samples. The sample collection should be done by appropriately trained personnel. Moreover, the abundance of the virus may vary depending on the stage of the infection. RT-qPCR studies have shown that the virus is abundant in the nasopharyngeal swabs during the initial stages of infection but can be detected in anal swabs also at later stages. However, the patient may become ELISA negative for NP samples at the later stages of infection. Therefore, the anatomical site of sample collection for antigen testing is determined by the stage of the infection.LFAs are supposed to be carried out at the point of care sites. Therefore, storage may not be a critical factor. If samples are transported it should be done under the guidelines of WHO. There is little information available on the effect of different storage conditions on the outcome of Ag LFAs for SARS-CoV-2 detection.The amount of sample being tested is a critical feature determining the sensitivity of any test. Sampling and sample application are considered as the Achilles heel of LFAs. In the case of the LFAs, the sample volume rarely exceeds 30 μL. Most often the LFAs are supplied as a kit with the dropper or other dispensing mechanism. The volume of the sample is the primary factor determining the flow rate along the membrane, which in turn determines the sensitivity and specificity of the test . Lack ofThe antibody used to detect antigen is the most critical part of the assay. The antibody used should have high differentiating ability and high affinity to the antigen. Generally monoclonal antibodies are used to increase specificity and avoid false positive reactions. The antibody chosen should be able to detect all variants of the virus. Manufacturers have not given any details regarding the antibodies used in the test .These tests are used to detect SARS-CoV-2 specific antibodies in the serum, blood or other biological fluids. The viral antigen conjugated with appropriate reporters such as colloidal gold, fluorescent/magnetic nanoparticle is impregnated in the membrane. Antibody present in the sample interacts with the conjugated antigen while moving through the membrane. LFA for detection of SARS-CoV-2 currently available in the market follows the broad pattern as shown in The number of test kits in the market is huge and the regulatory mechanisms in many countries are not adequate to safe guard from substandard kits . Early iThe antigen used is the most critical part of antibody detection assays. The nature of the antigen used in the commercial kits has not been revealed by many manufacturers. Sensitivity also varies with the antigens used for capturing the antibodies. Most often LFAs use partial proteins or peptides instead of whole antigens. When using such partial antigens many epitopes are lost, especially the conformational epitopes . TherefoOne of the major advantages of antibody detection LFA is avoidance of sampling error as liquid tissues such as blood, serum or plasma are used, which ensures the uniformity of the sample. Recent SARS-CoV-2 LFA kits use finger prick blood as the sample, which removes storage or transportation induced degradation of the samples. However, the person administering the test should be trained properly to avoid common mistakes like using clotted blood and suboptimal sample volume.Different antibody subtypes appear at different times post infection in an infected patient. IgM antibodies appear around 5–9 days post infection followed by IgG by days 10–14 . TherefoA new operator uses the kit;A new lot of test kits is used;A new shipment of kits is used;The temperature used during storage of the kit falls outside of 2–30 °C;The temperature of the test area falls outside of 15–30 °C;To verify a higher than expected frequency of positive or negative results;To investigate the cause of repeated invalid results;A new test environment is used .There can be a variation in sensitivity of the LFA kits based on the batch, transportation and storage. The representative samples should be evaluated with known positive and negative sera. Such quality evaluation should be done when:The most common LFA comes with a visual read out, eliminating the need of any equipment. Nevertheless, few drawbacks such as very limited sensitivity, individual variation in reading, inability to quantify and difficulty in electronic record keeping are associated with LFA. In order to overcome these drawbacks, assays are being made available using dedicated strip readers. The kits with strip reader facility employ colorimetric, fluorescent or magnetic reporters. The availability of fluorescent nanoparticles such as europium has hastened the adoption of this technique . These mData from the controls are essential to verify the accuracy and reliability of any diagnostic test. This is to ensure integrity and stability of reagents and robustness of the test process. ELISA and RT-qPCR have positive and negative controls incorporated, whereas no such controls are available in LFAs. The control line provides data regarding the optimum flow through the sample pad. Moreover, there is no control incorporated into each assay, which assures the stability of reagents. The lack of proper control is a major drawback of the LFA. ,82,83. SThe results of serological tests either positive or negative should be interpreted with care, taking into account the epidemiological factors in the play. Primarily, the record of onset of symptoms during which the samples were collected need to be available to the person deciding on the result. A false negative Ag or Ab tests can occur during the early stages of infection even if sampling errors are omitted. A false negative test can be provided by a test with low sensitivity, which is a case reported by many LFA test kits. False positive antibody reactions are a real possibility where previous SARS-CoV-1 outbreaks had been reported especially with kits using cross-reacting antigens like E gene protein.An antibody test provides limited information about the infection status of the individual. The earliest antibody reported is day five after onset of symptoms; however, the virus can be detected in nasal swabs even five days prior to the appearance of symptoms . TherefoThe LFAs or ELISA tests do not provide data regarding protective immunity of an individual. In general, LFAs or ELISA do not provide any information regarding the presence of virus neutralizing antibodies. In order to obtain such data, a virus neutralization test needs to be done, which is a laborious and time consuming test requiring specialized laboratories . MoreoveInadequate/improper sampling: Improper sampling technique may result in low amounts of biological specimen collected for RNA isolation, which may lead to false-negative results. CDC recommends synthetic fiber swabs with plastic or metal shafts . Swabs cImproper handling, storage and transport of samples: Swabs must be stored in containers with recommended Viral Transport Medium (VTM) free of PCR inhibitors. Storage or transport at improper temperatures will lead to degradation of the sample and lead to false-negative results .Improper processing of sputum: Sputum is collected as a specimen in some cases for testing, and improper liquefaction of the sputum has been attributed to false-negative results .Timing of the specimen collection after the onset of symptoms: The probability of detection of SARS-CoV-2 from NP/OP swabs by RT-qPCR alone decreases with the time since the onset of symptoms. The longer the time since the onset of symptoms until the case is tested, the more likely chance of false-negative results . In a reDifference in severity of infection: Individual differences in patients with regard to the severity of infection, viral load in the upper respiratory tracts may also account for the false negative test results .Taq DNA polymerase [PCR inhibitors: Common inhibitors in diagnostic PCRs include heme and humic acid. Antiviral drugs like Acyclovir also have been reported to inhibit lymerase . SamplesInexperienced laboratory staff and inadequate infrastructure in laboratories can lead to false positive results due to contamination and cross reactivity.False-negative antibody ELISA results: The sample used for antibody detection by ELISA is mostly blood. Blood being a uniform sample removes the inconsistency, which can occur with swab collection. However, care should be taken while selecting the collection tube, storage and transportation solutions. Blood being a nutrient-rich substance can get contaminated easily and it calls for ascetic handling while separating plasma or serum. It has been shown that blood samples contaminated with mold or bacteria give higher than normal OD in ELISA. Improper handling and storage can lead to hemolysis of blood. Even though ELISA is relatively immune to artifacts resulting from hemolysis, changes in final OD values have been recorded.This review compiles the available test platforms for the diagnosis of COVID-19, one of the most dreaded infections of mankind in the 21st century. The global scientific community is engraved in developing measures to contain this infection using a multitude of methodologies and diagnosis, which is the starting step in the process of controlling the contagion. The diagnosis has diverse magnitudes; at the individual level it deals with the prognosis of the patient, at the population level it provides the epidemiological surveillance data, and at the mass control level it speaks about the herd immunity. The presence of asymptomatic cases with potential to transmit the infection further stresses the importance of accurate diagnosis in real time. The ratio of asymptomatic infections to the symptomatic infection varies from study to study but comes in the range of almost 2–41% ,88,89. T


The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium. Muscle contraction and relaxation on the molecular level is achieved by the sliding movement of interdigitating thick filaments containing myosin and thin filaments containing actin in the sarcomere. Fundamentally, this process is driven by the cyclic interaction between myosin heads and actin filaments coupled with ATP hydrolysis and conformational changes of the myosin head MyBP-C by MYBPC2 on chromosome 19 and cardiac (c)MyBP-C by MYBPC3 on chromosome 11. cMyBP-C was discovered after ssMyBP-C and fsMyBP-C (Hartzell and Titus Myosin binding protein C (MyBP-C) is a sarcomeric accessory protein that was first identified as a contaminant of crude skeletal muscle preparation   targets on MyBP-C or inducing phosphorylation of the RLC by myosin light chain kinase , a highly expressed protein in skeletal and cardiac muscle. MyBP-C seems integral for the incorporation of FHL1 into the thick filament  with the cMyBP-C  and titin backbone  region of the thick filament and the actin filament.An important aspect of the way in which MyBP-C can regulate the thick filament (and the thin filament) is the 3-D geometrical packing of the proteins into the thick filament. There are 3 MyBP-C for each 9 myosins in the C-zone. Thus, the arrangement is important for how MyBP-C may directly or indirectly interact with each pair of myosin heads see Fig. . The 43 The functional effect of phosphorylation of the cMyBP-C M domain was first described in intact amphibian muscle Hartzell . Frog myMYBPC3 to suggest an interaction interface across one face of myosin (the ‘mesa’) in the ordered relaxed conformation  did show thin filament binding but no effect on the Ca2+ sensitivity and S1 binding  resulting in clear landing zones for these proteins, as demonstrated for myosin and actin using laser tweezers (Steffen et al. The more we seem to understand about cMyBP-C the more challenging a model is required to describe its function. We have summarised many of the observations in Fig. Despite these potential mechanisms of activation, other fundamental questions regarding the localisation of cMyBP-C in the C-zone also need to be addressed. The diversity of cMyBP-C binding partners might suggest a role in sarcomeric organisation and repair, or to offset the calcium gradient in the cardiac sarcomere (Previs et al. Finally, the molecular picture provided in this review provides a framework that will ultimately require explanation at the physiological contraction level. Such observations show inconsistencies between studies possibly due to the method of study or sample preparation which may affect lattice spacing (Irving and Craig The continuing story of cMyBP-C is providing a revelation in our understanding of muscle, both in normal and disease conditions. With the advancements in imaging and spectroscopy technologies across a range of resolutions both in vivo and in vitro it is hoped that these can be provide a detailed time-resolved view of cMyBP-C function. Reconciling a stronger molecular understanding with physiological observations using computational methods offers a mechanism to solve the remaining questions of how cMyBP-C modulates contraction and why it is such a prolific site of mutation in disease.


Dientamoeba, Entamoeba, Endolimax, Hymenolepis) that are often excluded from routine diagnostic protocols. The study clearly demonstrates the applicability of untargeted RNA metagenomics for the parallel detection of parasites.Shotgun metagenomics with high-throughput sequencing (HTS) techniques is increasingly used for pathogen identification and characterization. While many studies apply targeted amplicon sequencing, here we used untargeted metagenomics to simultaneously identify protists and helminths in pre-diagnosed faecal and tissue samples. The approach starts from RNA and operates without an amplification step, therefore allowing the detection of all eukaryotes, including pathogens, since it circumvents the bias typically observed in amplicon-based HTS approaches. The generated metagenomics datasets were analysed using the RIEMS tool for initial taxonomic read assignment. Mapping analyses against ribosomal reference sequences were subsequently applied to extract 18S rRNA sequences abundantly present in the sequence datasets. The original diagnosis, which was based on microscopy and/or PCR, could be confirmed in nearly all cases using ribosomal RNA metagenomics. In addition to the pre-diagnosed taxa, we detected other intestinal eukaryotic parasites of uncertain pathogenicity (of the genera The online version of this article (10.1007/s00436-020-06754-9) contains supplementary material, which is available to authorized users. Microscopy remains the gold standard for diagnosis of many parasitic infections, but requires skilled personnel and is time consuming. Moreover, morphologic features are often insufficient to identify the organism at the species level. Furthermore, the relatively low prevalence of many parasitic infections in Western industrialized countries has caused a decrease in awareness and a loss of expertise. Conventional molecular pathogen detection is also a challenge for emerging or neglected pathogens for which no sequence information and consequently no well-established test systems are available. The establishment of new specific tests is complicated by the lack of standardized reference material, especially in the case of parasites. For all these reasons, a fast and efficient one-serves-all approach is desirable for rapid and synchronous parasite detection.Untargeted (PCR-free) metagenomics approaches are well suited for the detection of pathogens of different phylogenetic affiliations including viral and bacterial pathogens  samples were microscopically checked before transfer to the FLI and the diagnoses were provided with the material. Upon arrival at FLI, all samples were stored deeply frozen (−80 °C) until further processing.Diagnostic stool samples from different archive collections were included in this study: 1) Ethanol-fixed parasite-containing stool samples were obtained from the Medical University of Vienna, Austria (MUV samples). The samples were pre-diagnosed by light microscopy after sodium acetate-acetic acid-ethanol concentration.  Ethanol-Ethanol-fixed samples were centrifuged  to remove ethanol, and the pellets were washed three times using 1 ml of 1 × TE buffer by mixing and subsequent centrifugation. The washed and re-suspended pellets (for MUV samples), the thawed ISS samples and pieces of the VD samples were treated with the cryoPREP (CP) instrument (Covaris) for disintegration as described  and a minimum overlap length of reads of 95% were applied for reference mapping. The obtained 18S rRNA sequences were verified by Blast analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Whereas for the proof-of-concept study . They are accessible under the accession numbers MN914072-MN914086.The 18S rRNA sequences generated within this study were submitted to GenBank when the fragments were longer than 1000 base pairs . The fixation of the MUV-4 sample might have had a negative impact on the recovery of RNA. The resulting dataset was therefore too small for a successful detection of the taxa. Since helminths can be distributed very unevenly in the host tissue and their eggs in stool samples, respectively, the reason for failure with sample VD-3 could be the use of a disadvantageous subsample not containing any specimen of the pre-diagnosed taxa. This might also be true for VD-2. The dataset contained only a few reads of the liver fluke  and 15.7% .An overview of samples and associated results is given in Table Blastocystis and Ascaris was higher without disintegration than with any disintegration. In these datasets, the number of parasite reads is especially influenced by shifts of the detected bacterial read numbers. As already shown, the application of a disintegration step may result in a higher representation of nucleic acids from specific certain bacteria . This is mainly caused by the amount of unclassified reads (Table For two samples (ISS-2 and ISS-6), sample processing with disintegration (CP and CP + M220 Focused-ultrasonicator) and without disintegration was compared. For sample ISS-2, the detected number of reads from ds Table  that wasds Table . This neds Table . While iCryptosporidium spp., Giardia spp.; Supplementary Table S1) extracted with the RNeasy Mini Kit compared to the datasets of the same samples extracted with the Agencourt RNAdvance Tissue Kit. Although the results for the two samples rather support the use of the RNeasy Mini Kit, the selection of the optimal kit needs to be investigated in a systematic validation and might depend on the sample type.With respect to the RNA extraction kits used, we could detect higher parasite read numbers for both samples ISS-C and ISS-D  as reference sequence. When using a full genome sequence (UZAE01000001) for reference mapping, we found that most of the detected reads match with the rRNA tandem repeat unit but not necessarily with the 18S rRNA gene. Therefore, we re-run the reference mapping using the ribosomal tandem repeat (X52949) as reference, which resulted in a better mapping outcome though the read amount was still very low (<0.0009%) and reads mostly belonged to the 28S rRNA gene that is longer than the 18S rRNA gene. The ribosomal sequences of Giardia exhibit a very high GC content  resulted in a very poor library delivering only a small dataset.Blastocystis was confirmed in all pre-diagnosed cases and additionally found in a sample that had been pre-screened only by light microscopy but not by PCR indicating a low Blastocystis density in the respective sample (MUV-1). We detected additional species using the metagenomics approach. Since these taxa cause no severe diseases, according to the current state of knowledge, they are typically not included in routine diagnostics. For example, Endolimax nana belongs to the family Entamoebidae and is often observed in faecal samples but, due to its non-pathogenic nature, it is typically not reported. Likewise, enteric infections with Hymenolepis nana and Dientamoeba fragilis are typically considered benign, although controversy persists regarding their pathogenic potential. For example, H. nana was identified as causative agent in a case of invasive cestodiasis in an immunocompromised patient with a spread of the infection to an ectopic location  are false-positive hits based on misassignments due to not correctly curated genomes in public databases or the lack of suitable reference genomes as already discussed in detail  occurred in some Blast analyses as closest hits to the query sequence. However, the mentioned sequences are closely related to Dipodascaceae (Ascomycota) and do not show any close relationship to Blastocystis species .Some implausible findings obtained with the RIEMS tool . Untargeted metagenomics sequencing is therefore applicable as one-serves-all pathogen detection tool swiftly providing comprehensive information of the sample. We recommend using it as a snapshot approach especially in cases with a difficult diagnosis because of contradictory symptoms or for infections in immunocompromised individuals. However, reasonable care has to be taken with the analysis of the obtained results using current public databases.Altogether, the application of untargeted RNA metagenomics sequencing for diagnostic samples was promising in the present study, especially since it can be used for the parallel identification of different unrelated taxa. In addition, it provides a more complete picture on protists that are often not included in routine diagnostics


Prokaryotic translation is among the major targets of diverse natural products with antibacterial activity including several classes of clinically relevant antibiotics. In this review, we summarize the information about the structure, biosynthesis, and modes of action of translation inhibiting ribosomally synthesized and post-translationally modified peptides (RiPPs). Azol(in)e-containing RiPPs are known to target translation, and several new compounds inhibiting the ribosome have been characterized recently. We performed a systematic search for biosynthetic gene clusters (BGCs) of azol(in)e-containing RiPPs. This search uncovered several groups of clusters that likely direct the synthesis of novel compounds, some of which may be targeting the ribosome. Antibiotics are extensively used worldwide in healthcare, agriculture, and food preservation. However, development and the spread of resistance to most antibiotics discovered during the second half of the 20th century in the course of the so-called “golden era of antibiotics” has become a global threat . With muin silico predictions of functions of the groups of genes called “biosynthetic gene clusters” (BGCs), whose products may take part in the biosynthesis of a certain metabolite. Making specific predictions about the structure of the final compound enables better prioritizing of candidate BGCs for subsequent time-consuming downstream experimental validation. Both proteinogenic and non-proteinogenic amino acids can act as building blocks for the production of specialized metabolites, leading to a great diversity of naturally occurring bioactive peptides. Peptide natural products originating from bacteria and fungi and currently used as antibiotics are dominated by non-ribosomal peptides (NRPs), assembled by large multisubunit enzymatic complexes  comprise another rapidly expanding class of bioactive peptides. They are produced by posttranslational modifications (PTMs) of ribosomally synthesized precursors by dedicated enzyme machinery . CompareAmong the RiPPs exhibiting antibacterial activity, there are examples of compounds targeting various validated drug targets, including bacterial RNA polymerase  and a conserved region e heterocycles, dehydration of amino acids, and macrocyclization e cycles , red arevia a glutamylation-elimination mechanism with tRNAGlu functioning as a donor of glutamyl (a to e)  .laz-cluster of lactazole synthesis (nos- and tbd-clusters) as well as transporters and regulatory proteins. The tailoring modifications may include the formation of a side ring system via the addition of indole derivates  have azol(in)e cycles installed along the polypeptide backbone and (ii) do not undergo macrocyclization (The name “linear azol(in)e-containing peptides (LAPs)” refers to the only two characteristics shared by compounds from this diverse subgroup of RiPPs: they  resemble a “sea with islands,” where each “island” is formed by a group of the closely related homologs , genes encoding the enzymes required for azole cycle installation (klpCBD), and an exporter pump gene (klpE)  is the first characterized translation-targeting LAP . Its BGCe (klpE) . In addie (klpE) , a modifydratase . The amiThermus thermophilus ribosome  revealed the molecular details of its mode of action. KLB binds in the upper part of the peptide exit tunnel in a site adjacent to the peptidyl-transferase center (PTC)  . Acting Rhizobium sp. Pop5, a symbiont of wild beans Phaseolus vulgaris (Escherichia coli ribosome complex with PHZ (T. thermophilus ribosome.Phazolicin (PHZ) is another recently discovered ribosome-targeting LAP produced by soil bacterium vulgaris . In termvulgaris . PHZ is vulgaris . PHZ is with PHZ . Four azEnterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA)  . In earl in vivo . Further in vivo , 1983 anStreptomyces bottropensis in 1957  is a peptide-nucleotide antibiotic produced by  cluster , which ehosphate . MccD anhosphate . Recent hosphate .via the inner membrane transporter YejABEF, which recognizes the peptide part of McC , which does not directly interact with the ribosome but blocks translation by inhibiting the supply of substrates required for protein synthesis. Although McC has been studied for more than 30 years and structures of multiple enzymes involved in its biosynthesis and immunity have been determined e cycles. At least in several cases where the mode of interaction with a ribosome is known, these cycles take part in stacking interactions with rRNA nucleobases thus mediating binding of the inhibitor to the target. We decided to perform a search for novel groups of azol(in)e-containing RiPPs in the genomes present in publicly available databases with a goal of identifying the putative translation inhibitors as well as other bioactive molecules. Due to their essential role in azol(in)e-containing RiPP biosynthesis, the genes encoding the YcaO-domain-containing enzymes were chosen as a starting point for our search.In less than 10 years YcaO-domain containing enzymes went from being DUFs (domains of unknown function) to one of the most studied groups of RiPP modification proteins . It was ycaO genes (lanB-like genes) as they were recently searched with various tools (To identify new BGCs of azol(in)e-containing compounds, we started with a sensitive search for sequences of YcaO domain-containing enzymes present in genomes from the RefSeq database . In brieaO genes . To visuaO genes  or descraO genes . In the us tools . The genin vitro, but the structure of the naturally produced compound remains unknown , includes three auxiliary genes . The product of gene D1 is the second YcaO protein. It is distinct from the product of the D2 gene and lacks the C-terminal PxP-motif, found in azoline-forming YcaOs and involved in catalysis (2 is distantly related to ThiF/MccB/PaaA proteins and contains a RiPP recognition element (RRE) — a domain found in different RiPP modification enzymes binding leader peptides   . McbG isin (PRP) . Clustert gyrase .mcb-operon homolog from P. syringae (blue circle). However, several of these clusters (marked with red letter G) contain a gene coding for a PRP protein. The overall sequence similarity and the distribution of potentially cyclizable residues in precursor peptides from clusters with and without the PRP gene differ  do affect translation.A relatively large network of clusters retrieved by our search , Group 2e differ . Thus, iAlgibacter aquaticus SK-16 . We hope that the upcoming years will provide more structural insights not only on the enzymology of RiPP modification widely studied now, but also on the principles the already known and novel compounds act by.In many cases, the analysis of genomic information was a starting point for further successful discoveries of a novel RiPP, facilitating the prediction of the BGC product based on the sequences of precursor peptides and modification enzymes. Through genome mining, future studies will not only result in the discovery of new compounds but will also allow systemization of our knowledge about RiPP genomic landscape and a better understanding of RiPP clusters’ evolutionary relations.1. We clustered resulting hits with mmseqs2  we converted RODEO output to genbank files imitating antiSMASH (Using a custom script (available on ntiSMASH  output. ntiSMASH  were marntiSMASH  subsequentiSMASH .DT and DB performed the bioinformatic search and interpreted its results. DT prepared the figures. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.