Comparative Genomic and Transcriptomic Profiling Revealed the Molecular Basis of Starch Promoting the Growth and Proliferation of Balantioides coli.

Carbohydrates are the main source of nutrition for B. coli, supplying energy for cell growth and development. The research aimed at investigating the mechanism of starch on the growth and replication of B. coli. Single-cell separation was used to isolate single trophozoites of B. coli under a stereomicroscope, transcriptomic profiling was conducted based on the SMART-seq2 single-cell RNA-seq method. Comparative genomic analysis was performed on B. coli and eight other ciliates to obtain specific and expanded gene families of B. coli. GO and KEGG enrichment analysis were used to analyze the key genes of B. coli under the action of starch in the present study. The results of single-cell RNA-seq depicts starch affected the growth and replication of B. coli in two ways: (1) the cell cycle was positively promoted by the activation of the cAMP/PKA signaling pathway via glycolysis; (2) the cell autophagy was suppressed through the PI3K/AKT/mTOR pathway. Genes involved in endocytosis, carbohydrate utilization, and the cAMP/PKA signaling pathway were highly enriched in both specific and expanded gene families of B. coli. Starch can be ingested and hydrolyzed into glucose, in turn affecting various biological processes of B. coli. The molecular mechanism of the effect of starch on the growth and proliferation of B. coli by promoting cell cycle and inhibiting the autophagy of trophozoites has been elucidated in our study.

Neospora caninum inhibits tumor development by activating the immune response and destroying tumor cells in a B16F10 melanoma model.

BACKGROUND: Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect of Neospora caninum, a livestock pathogen with no pathogenic activity in humans. METHODS: Neospora caninum tachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8+ T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect of N. caninum on gut microbial diversity. RESULTS: Intratumoral and distal subcutaneous injections of N. caninum resulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis. Neospora caninum treatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8+ T cells and macrophages in the TME was observed with these cytokine changes. Neospora caninum also restored the abundance of gut microbiota Lactobacillus, Lachnospiraceae, Adlercreutzia, and Prevotellaceae associated with tumor growth, but the changes were not significant. CONCLUSION: Neospora caninum inhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties of N. caninum demonstrate the potential for its use as a cancer treatment.

Neuritin-overexpressing transgenic mice demonstrate enhanced neuroregeneration capacity and improved spatial learning and memory recovery after ischemia-reperfusion injury.

Acute ischemia-reperfusion (IR)-induced brain injury is further exacerbated by a series of slower secondary pathogenic events, including delayed apoptosis due to neurotrophic factor deficiency. Neuritin, a neurotrophic factor regulating nervous system development and plasticity, is a potential therapeutic target for treatment of IR injury. In this study, Neuritin-overexpressing transgenic (Tg) mice were produced by pronuclear injection and offspring with high overexpression used to generate a line with stable inheritance for testing the neuroprotective capacity of Neuritin against transient global ischemia (TGI). Compared to wild-type mice, transgenic mice demonstrated reduced degradation of the DNA repair factor poly [ADP-ribose] polymerase 1 (PARP 1) in the hippocampus, indicating decreased hippocampal apoptosis rate, and a greater number of surviving hippocampal neurons during the first week post-TGI. In addition, Tg mice showed increased expression of the regeneration markers NF-200, synaptophysin, and GAP-43, and improved recovery of spatial learning and memory. Our findings exhibited that the window of opportunity of neural recovery in Neuritin transgenic mice group had a tendency to move ahead after TGI, which indicated that Neuritin can be used as a potential new therapeutic strategy for improving the outcome of cerebral ischemia injury.

Prevalence and molecular identification of Balantioides coli isolates from pet guinea pigs in Central China.

Balantioides coli is the only known zoonotic ciliate that can infect humans and is usually acquired from swine. It has, however, been reported in other mammals, including guinea pigs, where infection prevalence and molecular characterization are relatively unknown. In the present study, 32 guinea pigs from two different pet markets in Luoyang city of the Henan province in China were evaluated for ciliate-like trophozoites or cysts by direct fecal smear microscopy. Positive samples were further characterized using 18S rDNA and ITS1-5.8S rDNA-ITS2 sequence analysis. Microscopy indicated that ciliate-like cysts were observed in the fecal samples of several guinea pigs, were spherical in shape, and exhibited sizes of 40-65 µm in diameter. The average cyst-positive prevalence in guinea pigs was 62.5%. Sequence analysis indicated that the guinea pig-derived ciliate isolates belonged to B. coli and included two genetic variants (A and B), of which genetic variant A was more dominant among the guinea pig samples. To the best of our knowledge, the present study is the first molecular identification of B. coli in guinea pigs and provides some important information for investigating the molecular epidemiology of B. coli.

Molecular Detection and Genotyping of Toxoplasma gondii and Neospora caninum in Slaughtered Goats in Central China.

Toxoplasma gondii and Neospora caninum infections can cause reproductive failure in animals, including goats, and toxoplasmosis is one of the most important foodborne diseases. However, information on the molecular prevalence and genetic characterization of T. gondii and N. caninum in the tissues of goats in China is limited. In this study, brain samples of 422 slaughtered goats were collected from slaughterhouses in Henan and Anhui provinces, Central China, and examined for the presence of T. gondii and N. caninum by nested polymerase chain reaction (PCR) based on B1 and NC5 genes, respectively. The prevalence of T. gondii and N. caninum DNA was 5.2% and 2.8%, respectively. No significant differences were found between the prevalences of two parasite infections and animal age, sex, and region (p > 0.05). Two of 22 T. gondii-positive samples were completely genotyped at 11 genetic markers (SAG1, [3' + 5'] SAG2, alternative SAG2 [alt. SAG2], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using PCR-restriction fragment length polymorphism, and were identified as genotype ToxoDB no. 225, which has not been previously reported in goats in any country worldwide. For N. caninum, two different sequences at the ITS1 region, three genotypes at the MS5 microsatellite locus, and one genotype at the MS8 locus were identified. This study showed that T. gondii and N. caninum are moderately prevalent in goats in Central China; however, it should be emphasized that T. gondii prevalence in goats poses a potential health threat for consumers in the investigated areas. To the best of our knowledge, this is the first report on the genetic characterization of N. caninum isolates from goats in China. Our results have important implications for a better understanding of the genetic diversity of these parasites in China.

GRP94 regulates M1 macrophage polarization and insulin resistance.

Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.

Genomic organization of the chicken TCRß locus originated by duplication of a Vß segment combined with a trypsinogen gene.

Based on the latest assembly of the red jungle fowl (Gallus gallus) genome sequence, we characterized the detailed genomic organization of the T cell receptor beta (TCRß) locus of chicken. The chicken TCRß locus spans approximately 210 kb, and is organized in a typical translocon organization as previously reported. Within this locus, a total of 16 germline Vß gene segments were classified into three subgroups, containing 11, four, and one members, respectively. Phylogenetic analysis revealed that the chicken Vß3.1 segment was homologous with the duck Vß1 subgroup, and further clustered with Vß  segments from reptiles but not amphibians. We also identified nine protease serine 1 (PRSS1) and three protease serine 2 (PRSS2) genes, which were interspersed within the chicken TCRß locus. Dot-plot analysis of the chicken TCRß locus against itself revealed that the 5' part of the locus had arisen through a series of tandem duplication events. The homology units were composed of one Vß1 segment followed by a PRSS1 gene, or one Vß2 segment followed by a PRSS2 gene. This duplication pattern, in which the Vß segments and trypsinogen genes form a duplication unit, was unique to TCRß loci of chicken and duck, but not observed in TCRß loci of other tetrapods studied thus far. By analyzing the cloned TCRß cDNA sequences, we found that the usage pattern of Vß segments was consistent with the results of previous studies. These studies showed that members of the Vß1 subgroup are preferentially utilized in V-D-J recombination. Furthermore, we found that the Vß3.1 segment participated into V-D-J recombination, but at a very low frequency. The length distribution of the chicken complementarity-determining region 3ß (CDR3ß) showed a tendency similar to that observed for the duck.

Integrated mRNA-Seq and miRNA-Seq analysis of PLCγ2-overexpressing hepatocarcinoma cells and identification of the associated miRNA-mRNA network.

Our previous study has discovered the positive effect of phospholipase Cγ 2 (PLCγ2) on the growth of hepatocarcinoma cells; however, the underlying mechanism is far from being understood. For this reason, this study attempts to identify the differently expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in PLCγ2-overexpressing hepatocarcinoma cells. The results showed that totally 596 differently-expressed genes (DEGs) were identified in PLCγ2-expressed cells, including 314 upregulated and 282 downregulated ones; according to gene ontology analysis, these DEGs were involved in different cellular processes. Concurrently, 34 differently-expressed miRNAs (DEMs) were also detected in PLCγ2-expressing hepatocarcinoma cells. Moreover, the integrative analysis of miRNA and mRNA expression profiles identified the potential regulatory network linked to hepatocarcinoma-related biological processes, including metabolic activity, gene expression, cell cycle, cell migration, and so on. To our knowledge, it is the first study on the effect of PLCγ2 on miRNA and mRNA expressions in hepatocarcinoma cells, and the findings provide new insights into the mechanism supporting the growth-promoting effect of PLCγ2 in hepatocarcinoma cells.

Multi-stage constant-current charging protocol for a high-energy-density pouch cell based on a 622NCM/graphite system.

A novel multi-stage-constant-current (MS-CC) charging protocol, which charges high-energy-density lithium-ion cells (LICs) at a faster rate, is presented herein. In this work, the 0-80% state of charge (SoC), according to the maximum charging rate, yields acceptable results for different SoCs, and the charging process is divided into three parts. Twelve groups of experiments are designed under the desired conditions of avoiding lithium plating and using a charging time of less than 36 min, and 1.5C constant current charging is used as a comparison experiment. The full pouch cells are dismantled, and the lithium deposition after 1.5C charging is more extensive than that after the MS-CC charging protocol. In addition, the capacity retention for 1.5C charging is 95.7%, while those for the 12 MS-CC charging protocol groups are within the range of 99.5-100.0% after the 300th cycle at 25 °C. When the temperature is 25 °C and 50 °C, the capacity retention of the 12 MS-CC charging protocol groups remains similar, but when the temperature drops to 10 °C, the capacity retention decreases except for the 2.0-1.5-0.9C and 1.8-1.5-0.9C groups. At the 510th cycle, the capacity retention of the 2.0-1.5-0.9C and 1.8-1.5-0.9C groups is 99.6% and 99.9%, respectively; the values of the other 10 groups are between 95.0% and 98.2%. The excellent electrochemical performances of the MS-CC charging protocol may be due to the minimal damage of cell materials caused by the step-type high-rate charging process; thus, the degree of polarization is small. Furthermore, compared with the conventional constant constant-current (CC) charging procedure, MS-CC charging greatly shortens the charging time.

Prevalence and molecular characterization of Sarcocystis infections of retail beef products from central China.

Cattle are the intermediate hosts for five Sarcocystis species including S. hominis and S. heydorni, which also infect humans. To investigate the prevalence of Sarcocystis infections in beef products from 17 cities in the Henan Province of central China, 62 raw beef samples from markets were collected and analyzed for Sarcocystis presence via muscle squashing microscopic observation, histological section examination, and molecular characterization with 18S rRNA gene sequencing. Sarcocystis were detected in a total of 20 of the meat samples. Four species were identified that comprised S. cruzi, S. rommeli, S. heydorni, and S. hirsuta, with S. cruzi as the dominant species. In addition, seven of the 20 infected samples were infected with two or three species. Analysis of the 18S rRNA sequences recovered from these samples suggested very little genetic diversity within each species. This study represents the first molecular identification of Sarcocystis species infection in retail beef products from China. These findings will provide valuable information for evaluating the potential public health risk of bovine Sarcocystis species infections and the control of sarcocystosis in cattle.

First molecular identification of Buxtonella ciliates from captive-bred mangabeys (Cercocebus torquatus) from China.

Buxtonella species are large cyst-forming ciliates that infect ruminants and monkeys, and are morphologically similar to Balantidium coli ciliates that infect pigs, humans, monkeys, and other animals. In this study, we isolated spherical cysts of ciliates that were similar to those of Balantidium and Buxtonella species within collared mangabeys (Cercocebus torquatus) from the Wangcheng Zoo of Luoyang in the Henan Province of central China. The cysts were further identified and designated as belonging to the Buxtonella monkey genotype based on molecular analyses of 18S rRNA, 5.8S rRNA, and ITS1-5.8S rRNA-ITS2 genetic markers. To our knowledge, this is the first report of Buxtonella monkey genotype within monkeys in China. These results will help clarify the classification of species of cyst-forming ciliate infections in monkeys.

MiR-34a is Involved in the Decrease of ATP Contents Induced by Resistin Through Target on ATP5S in HepG2 Cells.

Resistin is associated with metabolic syndrome and deciphering its developmental and molecular mechanisms may help the development of new treatments. MiRNAs serve as negative regulators in many physiological and pathological processes. Here, miRNA microarrays were used to detect differences in expression between resistin-treated and control mice, and results showed miR-34a to be upregulated by resistin. The purpose of this study was to determine whether miR-34a played a role in resistin-induced decrease of ATP contents. Transient transfection of miR-34a mimics was used to overexpress miR-34a and quantitative RT-PCR was used to detect its expression. Western blot analysis was used to determine the rate of expression at the protein level. ATP content was measured using an ATP assay kit. The target gene of miR-34a was analyzed using bioinformatics and confirmed with dual-luciferase report system. MiR-34a was upregulated by resistin in HepG2 cells, and overexpression of miR-34a was found to diminish ATP levels significantly. This study is the first to show that ATP5S is one of the target genes of miR-34a. Resistin diminishes ATP content through the targeting of ATP5S mRNA 3'UTR by miR-34a.

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish.

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that "tetrapod-specific" Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.

Extensive diversification of IgH subclass-encoding genes and IgM subclass switching in crocodilians.

Crocodilians are a group of reptiles that are closely related to birds and are thought to possess a strong immune system. Here we report that the IgH locus in the Siamese crocodile and the Chinese alligator contains multiple µ genes, in contrast to other tetrapods. Both the µ2 and µ3 genes are expressed through class-switch recombination involving the switch region and germline transcription. Both IgM1 and IgM2 are present in the serum as polymers, which implies that IgM class switching may have significant roles in humoural immunity. The crocodilian α genes are the first IgA-encoding genes identified in reptiles, and these genes show an inverted transcriptional orientation similar to that of birds. The identification of both α and δ genes in crocodilians suggests that the IgH loci of modern living mammals, reptiles and birds share a common ancestral organization.

A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods.

In the past several decades, immunoglobulin (Ig) genes have been extensively characterized in many tetrapod species. This review focuses on the expressed Ig isotypes and the diversity of Ig genes in mammals, birds, reptiles, and amphibians. With regard to heavy chains, five Ig isotypes - IgM, IgD, IgG, IgA, and IgE - have been reported in mammals. Among these isotypes, IgM, IgD, and IgA (or its analog, IgX) are also found in non-mammalian tetrapods. Birds, reptiles, and amphibians express IgY, which is considered the precursor of IgG and IgE. Some species have developed unique isotypes of Ig, such as IgO in the platypus, IgF in Xenopus, and IgY (ΔFc) in ducks and turtles. The κ and λ light chains are both utilized in tetrapods, but the usage frequencies of κ and λ chains differ greatly among species. The diversity of Ig genes depends on several factors, including the germline repertoire and recombinatorial and post-recombinatorial diversity, and different species have evolved distinct mechanisms to generate antibody diversity.

Expressional analysis of immunoglobulin D in cattle (Bos taurus), a large domesticated ungulate.

For decades, it has remained unknown whether artiodactyls, such as cattle, pigs, and sheep, express immunoglobulin D (IgD), although the δ gene was identified in these species nearly 10 years ago. By developing a mouse anti-bovine IgD heavy chain monoclonal antibody (13C2), we show that secreted bovine IgD was present mainly as a monomer in serum and was heavily glycosylated by N-linked saccharides. Nonetheless, IgD was detectable in some but not all of the Holstein cattle examined. Membrane-bound IgD was detected in the spleen by western blotting. Flow cytometric analysis demonstrated that IgD-positive B cells constituted a much lower percentage of B cells in the bovine spleen (∼6.8% of total B cells), jejunal Peyer's patches (∼0.8%), and peripheral blood leukocytes (∼1.2%) than in humans and mice. Furthermore, IgD-positive B cells were almost undetectable in bovine bone marrow and ileal Peyer's patches. We also demonstrated that the bovine δ gene can be expressed via class switch recombination. Accordingly, bovine δ germline transcription, which involves an Iδ exon and is highly homologous to Iµ, was confirmed. However, we could not identify an Iδ promoter, despite bovine Eµ demonstrating both enhancer and promoter activity. This study has answered a long-standing question in cattle B cell biology and significantly contributes to our understanding of B cell development in this species.

Immunoglobulin genes and diversity: what we have learned from domestic animals.

This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species-cattle, sheep, pigs, and horses-express a full range of Ig heavy chains (IgHs), including µ, δ, γ, ϵ, and α. Two poultry species (chickens and ducks) express three IgH isotypes, µ, υ, and α, but not δ. The κ and λ light chains are both utilized in the four livestock species, but only the λ chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM), and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes.

Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single µ gene was identified, the α gene was not identified in this species.

Analysis of immunoglobulin transcripts in the ostrich Struthio camelus, a primitive avian species.

Previous studies on the immunoglobulin (Ig) genes in avian species are limited (mainly to galliformes and anseriformes) but have revealed several interesting features, including the absence of the IgD and Igκ encoding genes, inversion of the IgA encoding gene and the use of gene conversion as the primary mechanism to generate an antibody repertoire. To better understand the Ig genes and their evolutionary development in birds, we analyzed the Ig genes in the ostrich (Struthio camelus), which is one of the most primitive birds. Similar to the chicken and duck, the ostrich expressed only three IgH chain isotypes (IgM, IgA and IgY) and λ light chains. The IgM and IgY constant domains are similar to their counterparts described in other vertebrates. Although conventional IgM, IgA and IgY cDNAs were identified in the ostrich, we also detected a transcript encoding a short membrane-bound form of IgA (lacking the last two C(H) exons) that was undetectable at the protein level. No IgD or κ encoding genes were identified. The presence of a single leader peptide in the expressed heavy chain and light chain V regions indicates that gene conversion also plays a major role in the generation of antibody diversity in the ostrich. Because the ostrich is one of the most primitive living aves, this study suggests that the distinct features of the bird Ig genes appeared very early during the divergence of the avian species and are thus shared by most, if not all, avian species.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D.

Mutation of mevalonate kinase (MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome (HIDS) with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D (IgD) and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD (csIgD) specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.