Toxin Inhibition Research Articles

Dietary Metabolites and Chronic Kidney Disease.

Dietary contents and their metabolites are closely related to chronic kidney disease (CKD) progression. Advanced glycated end products (AGEs) are a type of uremic toxin produced by glycation. AGE accumulation is not only the result of elevated glucose levels or reduced renal clearance capacity, but it also promotes CKD progression. Indoxyl sulfate, another uremic toxin derived from amino acid metabolism, accumulates as CKD progresses and induces tubulointerstitial fibrosis and glomerular sclerosis. Specific types of amino acids (d-serine) or fatty acids (palmitate) are reported to be closely associated with CKD progression. Promising therapeutic targets associated with nutrition include uremic toxin absorbents and inhibitors of AGEs or the receptor for AGEs (RAGE). Probiotics and prebiotics maintain gut flora balance and also prevent CKD progression by enhancing gut barriers and reducing uremic toxin formation. Nrf2 signaling not only ameliorates oxidative stress but also reduces elevated AGE levels. Bardoxolone methyl, an Nrf2 activator and NF-κB suppressor, has been tested as a therapeutic agent, but the phase 3 clinical trial was terminated owing to the high rate of cardiovascular events. However, a phase 2 trial has been initiated in Japan, and the preliminary analysis reveals promising results without an increase in cardiovascular events.

Toxin Transport by A-B Type of Toxins in Eukaryotic Target Cells and Its Inhibition by Positively Charged Heterocyclic Molecules.

A-B types of toxins are among the most potent bacterial protein toxins produced by gram-positive bacteria. Prominent examples are the tripartite anthrax toxin of Bacillus anthracis and the different A-B type clostridial toxins that are the causative agents of severe human and animal diseases and could serve as biological weapons. The components of all these toxins comprise one binding/transport (B) subunit and one or two separate, non-linked enzymatically active (A) subunits. The A and B subunits are separately produced and secreted by the pathogenic gram-positive bacteria and must assemble on the surface of eukaryotic target cells to form biologically active toxin complexes. The B components are cleaved by proteases to generate the biologically active species that binds to receptors on the surface of the target cells and form there oligomers which bind the A subunits. The AB complexes are internalized by receptor-mediated endocytosis and reach early or late endosomes that become acidified. Subsequently, the B components form channels in endosomal membranes that are indispensable for the transport of the enzymatic subunits across these membranes into the cytosol of target cells via the trans-membrane channels. In addition to the channels formed by the B components, host cell factors including chaperones and further folding helper enzymes are involved in the import of the enzymatic subunits into the cytosol of eukaryotic cells. Positively charged heterocyclic molecules, such as chloroquine and related aminoquinolinium and azolopyridinium salts have been shown in recent years to bind with high affinity to the channels formed by the B components of binary toxins. Since binding to the B components is also a prerequisite for transport of the A components across the endosomal membranes the channel blockers also prevent transport of the A subunits into the host cell cytosol. The inhibition of toxin uptake into cells by such pharmacological compounds should also be of clinically interest because the toxins are the major virulence factors causing anthrax on the one hand and severe enteric disease on the other hand. Therefore, the novel toxin inhibitors should be attractive compounds for an application in combination with antibiotics to prevent or treat the diseases associated with binary toxins. Here the different processes involved in channel block in vitro and inhibition of intoxication of living target cells are reviewed in some detail.

A Cell-Based Fluorescent Assay to Detect the Activity of AB Toxins that Inhibit Protein Synthesis.

Many AB toxins elicit a cytotoxic effect involving the inhibition of protein synthesis. In this chapter, we describe a simple cell-based fluorescent assay to detect and quantify the inhibition of protein synthesis. The assay can also identify and characterize toxin inhibitors.

Low-Cost Charged-Coupled Device (CCD) Based Detectors for Shiga Toxins Activity Analysis.

To improve food safety there is a need to develop simple, low-cost sensitive devices for detection of food-borne pathogens and their toxins. We describe a simple, low-cost webcam-based detector which can be used for various optical detection modalities, including fluorescence, chemiluminescence, densitometry, and colorimetric assays. The portable battery-operated CCD-based detection system consists of four modules: (1) a webcam to measure and record light emission, (2) a sample plate to perform assays, (3) a light emitting diode (LED) for illumination, and (4) a portable computer to acquire and analyze images. To demonstrate the technology, we used a cell based assay for fluorescence detection of the activity of the food borne Shiga toxin type 2 (Stx2), differentiating between biologically active toxin and inactive toxin which is not a risk. The assay is based on Shiga toxin inhibition of cell protein synthesis measured through inhibition of the green fluorescent protein (GFP). In this assay, GFP emits light at 509nm when excited with a blue LED equipped with a filter at 486nm. The emitted light is then detected with a green filter at 535nm. Toxin activity is measured through a reduction in the 509nm emission. In this system the level of detection (LOD) for Stx2 was 0.1pg/ml, similar to the LOD of commercial fluorometers. These results demonstrate the utility and potential of low cost detectors for toxin activity. This approach could be readily adapted to the detection of other food-borne toxins.

Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding.

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo.

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

Chaperone addiction of toxin–antitoxin systems

Bacterial toxin–antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin–antitoxin–chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become ‘chaperone-addicted'. The chaperone specifically binds the antitoxin at a short carboxy-terminal sequence (chaperone addiction sequence, ChAD) that is not present in chaperone-independent antitoxins. In the absence of chaperone, the ChAD sequence destabilizes the antitoxin, thus preventing toxin inhibition. Chaperone–ChAD pairs can be transferred to classical TA systems or to unrelated proteins and render them chaperone-dependent. This mechanism might be used to optimize the expression and folding of heterologous proteins in bacterial hosts for biotechnological or medical purposes.

Alternative pre-approved and novel therapies for the treatment of anthrax.

Background Bacillus anthracis, the causative agent of anthrax, is a spore forming and toxin producing rod-shaped bacterium that is classified as a category A bioterror agent. This pathogenic microbe can be transmitted to both animals and humans. Clinical presentation depends on the route of entry (direct contact, ingestion, injection or aerosolization) with symptoms ranging from isolated skin infections to more severe manifestations such as cardiac or pulmonary shock, meningitis, and death. To date, anthrax is treatable if antibiotics are administered promptly and continued for 60 days. However, if treatment is delayed or administered improperly, the patient’s chances of survival are decreased drastically. In addition, antibiotics are ineffective against the harmful anthrax toxins and spores. Therefore, alternative therapeutics are essential. In this review article, we explore and discuss advances that have been made in anthrax therapy with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies.MethodsA literature search was conducted using the University of Manitoba search engine. Using this search engine allowed access to a greater variety of journals/articles that would have otherwise been restricted for general use. In order to be considered for discussion for this review, all articles must have been published later than 2009.ResultsThe alternative pre-approved antibiotics demonstrated high efficacy against B. anthracis both in vitro and in vivo. In addition, the safety profile and clinical pharmacology of these drugs were already known. Compounds that targeted underexploited bacterial processes (DNA replication, RNA synthesis, and cell division) were also very effective in combatting B. anthracis. In addition, these novel compounds prevented bacterial resistance. Targeting B. anthracis virulence, more specifically the anthrax toxins, increased the length of which treatment could be administered.ConclusionsSeveral novel and pre-existing antibiotics, as well as toxin inhibitors, have shown increasing promise. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.

VapC toxin inhibition as a method for prevention of the formation of resting forms of mycobacteria

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.

Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates.

Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and invitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane.

Varespladib (LY315920) Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation.

Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.

Deciphering the protein translation inhibition and coping mechanism of trichothecene toxin in resistant fungi

In modern times for combating the deleterious soil microbes for improved sustainable agricultural practices, there is a need to have a proper understanding of the plant-microbe interactions present in the rhizospheric microbiome of the plant roots. In the present study, the interactions of trichodermin with petidyltransferase centre of ribosomal complex was studied by molecular dynamics and in silico interaction methods to demonstrate its mechanism of action and to decipher the possible reason how it may inhibit protein synthesis at the ribosomal complex. Further we have illustrated how trichodermin resistance protein (60S ribosomal protein L3) helps to overcome the deleterious effects of trichothecene compounds like trichodermin. Normal mode analysis of trichodermin resistance protein and 25S rRNA that constitutes the petidyltransferase centre showed that the W-finger region of the protein moved towards 25S rRNA. Further analysis of molecular dynamics simulation time frames showed that several intermediate states of large motions of the protein molecules towards the 25S rRNA which finally blocks the binding pocket of the trichodermin. It indicated that this protein not only changes the local environment and conformation of the petidyltransferase centre but also restrain trichodermin from binding to the 25S rRNA at the petidyltransferase centre.

An update on antibody-based immunotherapies for Clostridium difficile infection.

Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review.

Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions.

The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from the trans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to the trans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potent in vitro inhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents.

Functional Characterization of Cholera Toxin Inhibitors Using Human Intestinal Organoids.

Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of IC50 values over a wide range of potencies (15 pM to 9 mM). The results indicate for the first time that an organoid-based swelling assay is a useful preclinical method to evaluate inhibitor potencies of drugs that target pathogen-derived toxins.

Synthetic multivalent ligands for cholera & cholera-like toxins: Protected cyclic neoglycopeptides

Synthesis of a set of novel glycopeptide analogues as potential cholera/cholera-like toxin inhibitors in their protected form is described. They include di-, tri-, tetra- and pentavalent scaffolds. The synthetic steps were achieved using a combination of solvent-free mechanochemical as well as the conventional solution-phase reactions. During the conventional DIC-HOBt-mediated peptide coupling followed for the preparation of certain glycopeptide analogues an interesting in situ Fmoc deprotection was observed which has been demonstrated to hold potential for synthesiszing glycopeptides/neoglycopeptides with extended polyamide chains.

Cholesterol regulates contractility and inotropic response to β2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein–Akt–NO-pathway

Majority of cardiac β2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-β-cyclodextrin (MβCD, 1 and 5mM) interferes with contractility and inotropic effect of β2-adrenergic agonist (fenoterol, 50μM) in the mouse atria. Treatment with MβCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MβCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MβCD, pertussis toxin or Akt inhibitor could recover the β2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MβCD-induced changes in the β2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to β2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the β2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B7) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B7 components of C2 toxin and LT, C2IIa and PA63, respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

DNA methylation profiling reveals differences in the 3 human monocyte subsets and identifies uremia to induce DNA methylation changes during differentiation

ABSTRACTHuman monocytes are a heterogeneous cell population consisting of 3 subsets: classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes. Via poorly characterized mechanisms, intermediate monocyte counts rise in chronic inflammatory diseases, among which chronic kidney disease is of particular epidemiologic importance. DNA methylation is a central epigenetic feature that controls hematopoiesis. By applying next-generation Methyl-Sequencing we now tested how far the 3 monocyte subsets differ in their DNA methylome and whether uremia induces DNA methylation changes in differentiating monocytes. We found that each monocyte subset displays a unique phenotype with regards to DNA methylation. Genes with differentially methylated promoter regions in intermediate monocytes were linked to distinct immunological processes, which is in line with results from recent gene expression analyses. In vitro, uremia induced dysregulation of DNA methylation in differentiating monocytes, which affected several transcription regulators important for monocyte differentiation (e.g., FLT3, HDAC1, MNT) and led to enhanced generation of intermediate monocytes. As potential mediator, the uremic toxin and methylation inhibitor S-adenosylhomocysteine induced shifts in monocyte subsets in vitro, and associated with monocyte subset counts in vivo. Our data support the concept of monocyte trichotomy and the distinct role of intermediate monocytes in human immunity. The shift in monocyte subsets that occurs in chronic kidney disease, a proinflammatory condition of substantial epidemiological impact, may be induced by accumulation of uremic toxins that mediate epigenetic dysregulation.

Novel Inhibitors of Toxin HipA Reduce Multidrug Tolerant Persisters

Persisters are a small fraction of drug-tolerant bacteria without any genotype variations. Their existence in many life-threatening infectious diseases presents a major challenge to antibiotic therapy. Persistence is highly related to toxin-antitoxin modules. HipA (high persistence A) was the first toxin found to contribute to Escherichia coli persistence. In this study, we used structure-based virtual screening for HipA inhibitors discovery and identified several novel inhibitors of HipA that remarkably reduced E. coli persistence. The most potent one decreased the persister fraction by more than five-fold with an in vitro K D of 270 ± 90 nM and an ex vivo EC50 of 46 ± 2 and 28 ± 1 μM for ampicillin and kanamycin screening, respectively. These findings demonstrated that inhibition of toxin can reduce bacterial persistence independent of the antibiotics used and provided a framework for persistence treatment by interfering with the toxin-antitoxin modules.