Identification and validation of circulating biomarkers for detection of liver cancer with antibody array.

The aim of this study was to find new protein biomarkers that could be used to detect hepatocellular carcinoma (HCC) in the serum. We identified 11 proteins in the tissue that could be used to classify samples from HCC and control subjects. The 11 identified tissue biomarkers were combined with 10 commonly used serum HCC biomarkers for further verification in a large number of serum samples from HCC patients and healthy controls. 17 of the 21 prospective serum biomarkers were determined to be differentially expressed through collinearity and significance analysis. Through the method of supervised learning, a random forest model was constructed to reduce the dimensionality of the number of differentially expressed proteins, and finally, 4 differentially expressed proteins were identified: AFP, GDF15, CEACAM-1, and MMP-9, and suggested to have potential application in clinical diagnosis of HCC.

Ionic Strength-Dependent Attachment of Pseudomonas aeruginosa PAO1 on Graphene Oxide Surfaces.

Graphene oxide (GO) is a widely used antimicrobial and antibiofouling material in surface modification. Although the antibacterial mechanisms of GO have been thoroughly elucidated, the dynamics of bacterial attachment on GO surfaces under environmentally relevant conditions remain largely unknown. In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to examine the dynamic attachment processes of a model organism Pseudomonas aeruginosa PAO1 onto GO surface under different ionic strengths (1-600 mM NaCl). Our results show the highest bacterial attachment at moderate ionic strengths (200-400 mM). The quantitative model of QCM-D reveals that the enhanced bacterial attachment is attributed to the higher contact area between bacterial cells and GO surface. The extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory and atomic force microscopy (AFM) analysis were employed to reveal the mechanisms of the bacteria-GO interactions under different ionic strengths. The strong electrostatic and steric repulsion at low ionic strengths (1-100 mM) was found to hinder the bacteria-GO interaction, while the limited polymer bridging caused by the collapse of biopolymer layers reduced cell attachment at a high ionic strength (600 mM). These findings advance our understanding of the ionic strength-dependent bacteria-GO interaction and provide implications to further improve the antibiofouling performance of GO-modified surfaces.

The Origin, Function, Distribution, Quantification, and Research Advances of Extracellular DNA.

In nature, DNA is ubiquitous, existing not only inside but also outside of the cells of organisms. Intracellular DNA (iDNA) plays an essential role in different stages of biological growth, and it is defined as the carrier of genetic information. In addition, extracellular DNA (eDNA) is not enclosed in living cells, accounting for a large proportion of total DNA in the environment. Both the lysis-dependent and lysis-independent pathways are involved in eDNA release, and the released DNA has diverse environmental functions. This review provides an insight into the origin as well as the multiple ecological functions of eDNA. Furthermore, the main research advancements of eDNA in the various ecological environments and the various model microorganisms are summarized. Furthermore, the major methods for eDNA extraction and quantification are evaluated.

Efficacy and safety of anlotinib in patients with advanced malignancy: a single-center, single-arm, phase 2 trial.

BACKGROUND: For advanced tumors that lack specific oncogenic alteration and are resistant to chemotherapy, anti-angiogenesis therapy or immunotherapy or a combination of the two are the most important treatments. Anlotinib is a newly developed oral small molecule receptor tyrosine kinases inhibitor with the potency of inhibiting tumor angiogenesis. This was an open-label, single-arm, phase 2 study to validate the efficacy and safety of anlotinib in patients with various cancer types. METHODS: Patients with advanced malignancy who have failed previous therapies or lack effective treatment choices received daily oral administration of 12 mg anlotinib on days 1-14 every 3 weeks until disease progression, intolerable toxicity or physician decision. The primary endpoint was objective response rate (ORR). RESULTS: A total of 93 eligible patients with 26 different cancer types were enrolled. The overall ORR was 21.5%. The median PFS was 5.7 months and median OS was 12.0 months. The most common treatment-related AE of all grades and of grade 3 was both hypertriglyceridemia at an incidence of 40.9% and 5.4%, respectively. CONCLUSIONS: Anlotinib exhibits objective efficacy and safety in advanced malignancy and might be a possible treatment option for many types of cancer patients who have failed prior treatment and with no optimal therapy regimen.

Outer Membrane c-Type Cytochromes OmcA and MtrC Play Distinct Roles in Enhancing the Attachment of Shewanella oneidensis MR-1 Cells to Goethite.

The outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC in Shewanella are key terminal reductases that bind and transfer electrons directly to iron (hydr)oxides. Although the amounts of OmcA and MtrC at the cell surface and their molecular structures are largely comparable, MtrC is known to play a more important role in dissimilatory iron reduction. To explore the roles of these outer membrane c-Cyts in the interaction of Shewanella oneidensis MR-1 with iron oxides, the processes of attachment of S. oneidensis MR-1 wild type and c-type cytochrome-deficient mutants (the ΔomcA, ΔmtrC, and ΔomcA ΔmtrC mutants) to goethite are compared via quartz crystal microbalance with dissipation monitoring (QCM-D). Strains with OmcA exhibit a rapid initial attachment. The quantitative model for QCM-D responses reveals that MtrC enhances the contact area and contact elasticity of cells with goethite by more than one and two times, respectively. In situ attenuated total reflectance Fourier transform infrared two-dimensional correlation spectroscopic (ATR-FTIR 2D-CoS) analysis shows that MtrC promotes the initial interfacial reaction via an inner-sphere coordination. Atomic force microscopy (AFM) analysis demonstrates that OmcA enhances the attractive force between cells and goethite by about 60%. As a result, OmcA contributes to a higher attractive force with goethite and induces a rapid short-term attachment, while MtrC is more important in the longer-term interaction through an enhanced contact area, which promotes interfacial reactions. These results reveal that c-Cyts OmcA and MtrC adopt different mechanisms for enhancing the attachment of S. oneidensis MR-1 cells to goethite. It improves our understanding of the function of outer membrane c-Cyts and the influence of cell surface macromolecules in cell-mineral interactions.IMPORTANCEShewanella species are one group of versatile and widespread dissimilatory iron-reducing bacteria, which are capable of respiring insoluble iron minerals via six multiheme c-type cytochromes. Outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC are the terminal reductases in this pathway and have comparable protein structures. In this study, we elucidate the different roles of OmcA and MtrC in the interaction of S. oneidensis MR-1 with goethite at the whole-cell level. OmcA confers enhanced affinity toward goethite and results in rapid attachment. Meanwhile, MtrC significantly increases the contact area of bacterial cells with goethite and promotes the interfacial reaction, which may explain its central role in extracellular electron transfer. This study provides novel insights into the role of bacterial surface macromolecules in the interfacial interaction of bacteria with minerals, which is critical to the development of a comprehensive understanding of cell-mineral interactions.

The exopolysaccharide-eDNA interaction modulates 3D architecture of Bacillus subtilis biofilm.

BACKGROUND: Bacterial biofilms are surface-adherent microbial communities in which individual cells are surrounded by a self-produced extracellular matrix of polysaccharides, extracellular DNA (eDNA) and proteins. Interactions among matrix components within biofilms are responsible for creating an adaptable structure during biofilm development. However, it is unclear how the interactions among matrix components contribute to the construction of the three-dimensional (3D) biofilm architecture. RESULTS: DNase I treatment significantly inhibited Bacillus subtilis biofilm formation in the early phases of biofilm development. Confocal laser scanning microscopy (CLSM) and image analysis revealed that eDNA was cooperative with exopolysaccharide (EPS) in the early stages of B. subtilis biofilm development, while EPS played a major structural role in the later stages. In addition, deletion of the EPS production gene epsG in B. subtilis SBE1 resulted in loss of the interaction between EPS and eDNA and reduced the biofilm biomass in pellicles at the air-liquid interface. The physical interaction between these two essential biofilm matrix components was confirmed by isothermal titration calorimetry (ITC). CONCLUSIONS: Biofilm 3D structures become interconnected through surrounding eDNA and EPS. eDNA interacts with EPS in the early phases of biofilm development, while EPS mainly participates in the maturation of biofilms. The findings of this study provide a better understanding of the role of the interaction between eDNA and EPS in shaping the biofilm 3D matrix structure and biofilm formation.

Divergent Influence to a Pathogen Invader by Resident Bacteria with Different Social Interactions.

Bacterial social interaction is a potential influencing factor in determining the fate of invading pathogens in diverse environments. In this study, interactions between two representative resident species (Bacillus subtilis and Pseudomonas putida) and a leading food-borne disease causative pathogen (Vibrio parahaemolyticus) were examined. An antagonistic effect toward V. parahaemolyticus was observed for B. subtilis but not for P. putida. However, the relative richness of the pathogen remained rather high in B. subtilis co-cultures and was, unexpectedly, not sensitive to the initial inoculation ratios. Furthermore, two approaches were found to be efficient at modulating the relative richness of the pathogen. (1) The addition of trace glycerol and manganese to Luria-Bertani medium (LBGM) reduced the richness of V. parahaemolyticus in the co-culture with B. subtilis and in contrast, increased its richness in the co-culture with P. putida, although it did not affect the growth of V. parahaemolyticus by its own. (2) The relative richness of V. parahaemolyticus on semisolid medium decreased significantly as a function of an agar gradient, ranging from 0 to 2%. Furthermore, we explored the molecular basis of bacterial interaction through transcriptomic analysis. In summary, we investigated the interactions between a pathogen invader and two resident bacteria species, showing that the different influences on a pathogen by different types of interactions can be modulated by chemicals and medium fluidity.

[Preparation and quality standard of standard decoction of Scrophulariae Radix pieces].

The standard decoction of Chinese herbal decoction pieces is a standard reference substance to measure whether different dosage forms of Chinese medicine are basically consistent with those of clinical decoction,and provides new ideas and methods for effectively solving the problems of uneven quality in Chinese medicine dispensing granules. In this study,a systematic method for evaluating the quality of Scrophulariae Radix decoction was established from the perspective of " standard decoction",providing reference for the quality control of the Scrophulariae Radix dispensing granules. 15 batches of Scrophulariae Radix decoction pieces from different origins were collected,and 15 batches of standard decoctions were prepared according to the standardized process with water as solvent.Harpagide and harpagoside were used as quantitative detection indicators to determine the content,calculate the transfer rates and determine the extraction rate. The high performance liquid chromatography( HPLC) was used to establish a standard decoction fingerprint analysis method. The results showed that the transfer rates of harpagide and harpagoside in 15 batches of Scrophulariae Radix pieces standard decoction were( 70. 84±13. 39) % and( 48. 56±6. 40) % respectively; the extraction rate was( 57. 47±5. 89) %. Nine peaks were identified in the HPLC fingerprint,and the similarity was higher than 0. 97 between the fingerprints of 15 batches of standard decoction and the control fingerprint. In this study,the preparation process of standard decoction of Scrophulariae Radix pieces conformed to the traditional decoction preparation method. The sources of the samples were representative,and the established fingerprint method was stable and feasible,which can provide reference for the preparation and quality control of Scrophulariae Radix dispensing granules.

The impact of advanced proteomics in the search for markers and therapeutic targets of bladder cancer.

Bladder cancer is the most common cancer of the urinary tract and can be avoided through proper surveillance and monitoring. Several genetic factors are known to contribute to the progression of bladder cancer, many of which produce molecules that serve as cancer biomarkers. Blood, urine, and tissue are commonly analyzed for the presence of biomarkers, which can be derived from either the nucleus or the mitochondria. Recent advances in proteomics have facilitated the high-throughput profiling of data generated from bladder cancer-related proteins or peptides in parallel with high sensitivity and specificity, providing a wealth of information for biomarker discovery and validation. However, the transmission of screening results from one laboratory to another remains the main disadvantage of these methods, a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization. This review summarizes the latest discoveries and progress of biomarker identification for the early diagnosis, projected prognosis, and therapeutic response of bladder cancer, informs the readers of the current status of proteomic-based biomarker findings, and suggests avenues for future work.

Metal-free inactivation of E. coli O157:H7 by fullerene/C3N4 hybrid under visible light irradiation.

Interest has grown in developing safe and high-performance photocatalysts based on metal-free materials for disinfection of bacterial pathogens under visible light irradiation. In this paper, the C60/C3N4 and C70/C3N4 hybrids were synthesized by a hydrothermal method, and characterized by X-ray diffraction (XRD), UV-vis diffuse reflection spectroscopy (UV-vis DRS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and high revolution transmission electron microscope (HRTEM). The performance of photocatalytic disinfection was investigated by the inactivation of Escherichia coli O157:H7. Both C60/C3N4 and C70/C3N4 hybrids showed similar crystalline structure and morphology with C3N4; however, the two composites exhibited stronger bacterial inactivation than C3N4. In particular, C70/C3N4 showed the highest bactericidal efficiency and was detrimental to all E. coli O157:H7 in 4h irradiation. Compared to C3N4, the enhancement of photocatalytic activity of composites could be attributed to the effective transfer of the photoinduced electrons under visible light irradiation. Owing to the excellent performance of fullerenes (C60, C70)/C3N4 composites, a visible light response and environmental friendly photocatalysts for disinfection were achieved.

Identification and functional study of type III-A CRISPR-Cas systems in clinical isolates of Staphylococcus aureus.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR associated proteins [Cas]) system can provide prokaryote with immunity against invading mobile genetic elements (MGEs) such as phages and plasmids, which are the main sources of staphylococcal accessory genes. To date, only a few Staphylococcus aureus strains containing CRISPR-Cas systems have been identified, but no functional study in these strains has been reported. In this study, 6 clinical isolates of S. aureus with type III-A CRISPR-Cas systems were identified, and whole-genome sequencing and functional study were conducted subsequently. Genome sequence analysis revealed a close linkage between the CRISPR-Cas system and the staphylococcal cassette chromosome mec (SCCmec) element in five strains. Comparative sequence analysis showed that the type III-A repeats are conserved within staphylococci, despite of the decreased conservation in trailer-end repeats. Highly homologous sequences of some spacers were identified in staphylococcal MGEs, and partially complementary sequences of spacers were mostly found in the coding strand of lytic regions in staphylococcal phages. Transformation experiments showed that S. aureus type III-A CRISPR-Cas system can specifically prevent plasmid transfer in a transcription-dependent manner. Base paring between crRNA and target sequence, the endoribonuclease, and the Csm complex were proved to be necessary for type III-A CRISPR-Cas immunity.

A TetR-like regulator broadly affects the expressions of diverse genes in Mycobacterium smegmatis.

Transcriptional regulation plays a critical role in the life cycle of Mycobacterium smegmatis and its related species, M. tuberculosis, the causative microbe for tuberculosis. However, the key transcriptional factors involved in broad regulation of diverse genes remain to be characterized in mycobacteria. In the present study, a TetR-like family transcriptional factor, Ms6564, was characterized in M. smegmatis as a master regulator. A conserved 19 bp-palindromic motif was identified for Ms6564 binding using DNaseI footprinting and EMSA. A total of 339 potential target genes for Ms6564 were further characterized by searching the M. smegmatis genome based on the sequence motif. Notably, Ms6564 bound with the promoters of 37 cell cycle and DNA damage/repair genes and regulated positively their expressions. The Ms6564-overexpressed recombinant strain yielded 5-fold lower mutation rates and mutation frequencies, whereas deletion of Ms6564 resulted in ∼5-fold higher mutation rates for the mutant strain compared with the wild-type strain. These findings suggested that Ms6564 may function as a global regulator and might be a sensor necessary for activation of DNA damage/repair genes.

Nanoemulsion for solubilization, stabilization, and in vitro release of pterostilbene for oral delivery.

Pterostilbene, being extracted from many plants, has significant biological activities in preventing cancer, diabetes, and cardiovascular diseases so as to have great potential applications in pharmaceutical fields. But the poor solubility and stability of pterostilbene strictly restrained its applications. As a good protection and oral delivery system, an optimal nanoemulsion for pterostilbene was developed by using low-energy emulsification method. Systematic pseudo-ternary phase diagrams have been studied in optimization of nanoemulsion formulations. The prepared pterostilbene nanoemulsion was characterized by transmission electron microscope, Fourier transform Raman spectrum, and laser droplet size analyzer. Nanoemulsion droplets are circular with smooth margin, and the mean size is 55.8 ± 10.5 nm. The results illustrated that the nanoemulsion as oral delivery system dramatically improved the stability and solubility of pterostilbene, and in vitro release of pterostilbene was significantly improved (96.5% in pH 3.6 buffer; 13.2% in pH 7.4 buffer) in comparison to the pterostilbene suspension (lower than 21.4% in pH 3.6 buffer; 2.6% in pH 7.4 buffer).

Underlying anti-cancer mechanisms of histone deacetylase (HDAC) inhibitors in tamoxifen-resistant breast cancer cells.

Objectives: Breast cancer is an important women's malignancy with high cancer-related deaths worldwide. Drug resistance lowers the treatment efficacy in this malignancy. This study aimed to explore the underlying mechanisms of histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to overcome resistance to tamoxifen in breast cancer cells. Materials and Methods: Tamoxifen-resistance in MCF-7 breast cancer cells was simulated. MTT assay was used to detect the cytotoxic effects of HDAC inhibitor and PI3K inhibitor on the cancer cells. Trans-well assay was applied to evaluate the invasion and migration of the treated cancer cells. Flow cytometer assay was also applied to evaluate cell cycle phases in the treated cancer cells. Finally, expression of vascular endothelial growth factor (VEGF), E-cadherin, Vimentin, phosphorylated phosphatidylinositol kinase (p-PI3k), phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target protein of rapamycin (p-mTOR) was evaluated by western blotting. Results: The obtained results indicated that HDAC inhibitor treatments significantly decreased viability, migration, and invasion in the cancer cells. Furthermore, the frequency of the treated cancer cells significantly increased in the S phase as well as significantly decreasing in the G2/M phase of the cell cycle. Moreover, HDAC inhibitor modified levels of VEGF, E-cadherin, Vimentin, p-PI3k, p-AKT, and p-mTOR proteins. However, HDAC inhibitor combined with PI3K inhibitor exerts more profound effects on the cancer cells as compared to HDAC inhibitor monotherapy. Conclusion: HDAC inhibitors inhibited the survival of breast cancer drug-resistant cells, invasion, migration, and angiogenesis by inhibiting the PI3k/Akt/mTOR signaling pathway.

ggVennDiagram: Intuitive Venn diagram software extended.

Highlights of ggVennDiagram include: (1) Subset/Region filling Venn diagram up to seven sets; (2) Upset plot with unlimited sets; (3) Venn Calculator for two or more sets; (4) Provide as R package, Shiny App, and TBtools plugin.

SdrR, a LysR-type regulator, responds to the mycobacterial antioxidant defense.

Protection against oxidative stress is a vital defense mechanism for Mycobacterium tuberculosis within the host. However, few transcription factors that control bacterial antioxidant defense are known. Here, we present evidence that SdrR, encoded by the MSMEG_5712 (Ms5712) gene, functions as an oxidative stress response regulator in Mycobacterium smegmatis. SdrR recognizes an 11-bp motif sequence in the operon's upstream regulatory region and negatively regulates the expression of short-chain dehydrogenases/reductases (SDR). Overexpressing sdrR inhibited SDR expression, which rendered the strain oxidative more stress-sensitive. Conversely, sdrR knockout alleviates SDR repression, which increases its oxidative stress tolerance. Thus, SdrR responds to oxidative stress by negatively regulating sdr expression. Therefore, this study elucidated an underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil.

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

The state and consideration for skin test of ß-lactam antibiotics in pediatrics.

ß-lactam antibiotics are the most frequently used drugs and the most common drugs that cause allergic reactions in pediatrics. The occurrence of some allergic reactions can be predicted by skin testing, especially severe adverse reactions such as anaphylactic shock. Thus, penicillin and cephalosporin skin tests are widely used to predict allergic reactions before medication in pediatrics. However, false-positive results from skin tests were more often encountered in pediatrics than in adults. In fact, many children labeled as allergic to ß-lactam are not allergic to the antibiotic, leading to the use of alternative antibiotics, which are less effective and more toxic, and the increase of antibiotic resistance. There has been controversy over whether ß-lactam antibiotics should be tested for skin allergies before application in children. Based on the great controversy in the implementation of ß-lactam antibiotic skin tests, especially the controversial cephalosporin skin tests in pediatrics, the mechanism and reasons of anaphylaxis to ß-lactam antibiotics, the significance of ß-lactam antibiotic skin tests, the current state of ß-lactam antibiotic skin tests at home and abroad, and the problems of domestic and international skin tests were analyzed to determine a unified standard of ß-lactam antibiotic skin tests in pediatrics to prevent and decrease adverse drug reactions, avoid waste of drugs, and a large amount of manpower and material resource consumption.

Extracellular polymeric substances and mineral interfacial reactions control the simultaneous immobilization and reduction of arsenic (As(V)).

Extracellular polymeric substances (EPS) play a crucial role in controlling the mobility and bioavailability of heavy metal(loid)s in water, soils, and sediments. The formation of EPS-mineral complex changes the reactivity of the end-member materials. However, little is known about the adsorption and redox mechanisms of arsenate (As(V)) in EPS and EPS-mineral complexes. Here we examined the reaction sites, valence state, thermodynamic parameters and distribution of As in the complexes using potentiometric titration, isothermal titration calorimetry (ITC), FTIR, XPS, and SEM-EDS. The results showed that ∼54% of As(V) was reduced to As(III) by EPS, potentially driven by an enthalpy change (ΔH) of - 24.95 kJ/mol. The EPS coating on minerals clearly affected the reactivity to As(V). The strong masking of functional sites between EPS and goethite inhibited both the adsorption and reduction of As. In contrast, the weak binding of EPS onto montmorillonite retained more reactive sites for the reaction with As. Meanwhile, montmorillonite facilitated the immobilization of As to EPS through the formation of As-organic bounds. Our findings deepen the understanding of EPS-mineral interfacial reactions in controlling the redox and mobility of As, and the knowledge is important for predicting the behavior of As in natural environments.

Regulation of CRISPR-Associated Genes by Rv1776c (CasR) in Mycobacterium tuberculosis.

The CRISPR-Cas system is an adaptive immune system for many bacteria and archaea to defend against foreign nucleic acid invasion, and this system is conserved in the genome of M. tuberculosis (Mtb). Although the CRISPR-Cas system-mediated immune defense mechanism has been revealed in Mtb, the regulation of cas gene expression is poorly understood. In this study, we identified a transcription factor, CasR (CRISPR-associated protein repressor, encoded by Rv1776c), and it could bind to the upstream DNA sequence of the CRISPR-Cas gene cluster and regulate the expression of cas genes. EMSA and ChIP assays confirmed that CasR could interact with the upstream sequence of the csm6 promoter, both in vivo and in vitro. Furthermore, DNA footprinting assay revealed that CasR recognized a 20 bp palindromic sequence motif and negatively regulated the expression of csm6. In conclusion, our research elucidates the regulatory effect of CasR on the expression of CRISPR-associated genes in mycobacteria, thus providing insight into gene expression regulation of the CRISPR-Cas system.