POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Structure of Telomerase with Telomeric DNA.

Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA. The catalytic core is an intricately interlocked structure of TERT and TER, including a previously structurally uncharacterized TERT domain that interacts with the TEN domain to physically enclose TER and regulate activity. This complete structure of a telomerase catalytic core and its interactions with telomeric DNA from the template to telomere-interacting p50-TEB complex provides unanticipated insights into telomerase assembly and catalytic cycle and a new paradigm for a reverse transcriptase RNP.

CST-Polα/Primase: the second telomere maintenance machine.

It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5' ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1-Stn1-Ten1 (CST). CST-Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5' ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST-Polα/Primase, highlighting the role of this complex and telomere length control in human disease.

53BP1: a DSB escort.

53BP1 is an enigmatic DNA damage response factor that gained prominence because it determines the efficacy of PARP1 inhibitory drugs (PARPi) in BRCA1-deficient cancers. Recent studies have elevated 53BP1 from its modest status of (yet another) DNA damage factor to master regulator of double-strand break (DSB) repair pathway choice. Our review of the literature suggests an alternative view. We propose that 53BP1 has evolved to avoid mutagenic repair outcomes and does so by controlling the processing of DNA ends and the dynamics of DSBs. The consequences of 53BP1 deficiency, such as diminished PARPi efficacy in BRCA1-deficient cells and altered repair of damaged telomeres, can be explained from this viewpoint. We further propose that some of the fidelity functions of 53BP1 coevolved with class switch recombination (CSR) in the immune system. We speculate that, rather than being deterministic in DSB repair pathway choice, 53BP1 functions as a DSB escort that guards against illegitimate and potentially tumorigenic recombination.

Models for human telomere C-strand fill-in by CST-Polα-primase.

Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA.

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5' DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA-binding ability of CST is required for blocking MRE11-mediated nascent-strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent-strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.

Cooperative interaction of CST and RECQ4 resolves G-quadruplexes and maintains telomere stability.

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.

Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

A Compact Linear Microstrip Patch Beamformer Antenna Array for Millimeter-Wave Future Communication.

5/6G is anticipated to address challenges such as low data speed and high latency in current cellular networks, particularly as the number of users overwhelms 4G and LTE capabilities. This paper proposes a microstrip patch antenna array comprising six radiating patches and utilizing a microstrip line feeding technique to facilitate the compact design crucial for 5G implementation. ROGER 3003, chosen for its advanced and environmentally friendly features, serves as the dielectric material, ensuring suitability for 5G and B5G applications. The designed antenna, evaluated at a resonating frequency of 28.8 GHz with a -10 dB impedance bandwidth of 1 GHz, offers a high gain of 9.19 dBi. Its compact array, cost-effectiveness, and broad impedance and radiation coverage position it as a viable candidate for 5G and future communication applications.

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus.

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1(CP)) in two siblings with CP. POT1(CP)induced a proliferative arrest that could be bypassed by telomerase. POT1(CP)was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1(CP)was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1(CP)was also defective in the maintenance of the telomeric C strand, causing extended 3' overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).

A Nested PCR Telomere Fusion Assay Highlights the Widespread End-Capping Protection of Arabidopsis CTC1.

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage-fusion-bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1.

CST2 promotes cell proliferation and regulates cell cycle by activating Wnt-ß-catenin signalling pathway in serous ovarian cancer.

BACKGROUND: Cystatin SA (CST2) plays multiple roles in different types of malignant tumours; however, its role in serous ovarian cancer (SOC) remains unclear. Therefore, we aimed to investigate the expression levels, survival outcomes, immune cell infiltration, proliferation, cell cycle, and underlying molecular mechanisms associated with the CST2 signature in SOC. METHODS: The Cancer Genome Atlas database was used to acquire clinical information and CST2 expression profiles from patients with SOC. Wilcoxon rank-sum tests were used to compare CST2 expression levels between SOC and normal ovarian tissues. A prognostic assessment of CST2 was conducted using Cox regression analysis and the Kaplan-Meier method. Differentially expressed genes were identified using functional enrichment analysis. Immune cell infiltration was examined using a single-sample gene set enrichment analysis. Cell cycle characteristics and proliferation were assessed using a colony formation assay, flow cytometry, and a cell counting kit-8 assay. Western blots and quantitative reverse transcription PCR analyses were employed to examine CST2 expressions and related genes involved in the cell cycle and the Wnt-ß-catenin signalling pathway. RESULTS: Our findings revealed significant upregulation of CST2 in SOC, and elevated CST2 expression was correlated with advanced clinicopathological characteristics and unfavourable prognoses. Pathway enrichment analysis highlighted the association between the cell cycle and the Wnt signalling pathway. Moreover, increased CST2 levels were positively correlated with immune cell infiltration. Functionally, CST2 played vital roles in promoting cell proliferation, orchestrating the G1-to-S phase transition, and driving malignant SOC progression through activating the Wnt-ß-catenin signalling pathway. CONCLUSIONS: The elevated expression of CST2 may be related to the occurrence and progression of SOC by activating the Wnt-ß-catenin pathway. Additionally, our findings suggest that CST2 is a promising novel biomarker with potential applications in therapeutic, prognostic, and diagnostic strategies for SOC.

Serous ovarian cancer is a type of gynecological malignant tumour with high mortality rates. Understanding this disease is crucial for improving treatments and enhancing patient survival. In our study, we investigated a protein called CST2 and its role in serous ovarian cancer. We found that CST2 levels vary among patients and are associated with the progression of cancer and the prognosis of the patient, which could be valuable for future diagnosis and treatment strategies. However, further research is needed to validate these findings. Despite its limitations, our findings suggest that CST2 holds promise as a potential biomarker for detecting serous ovarian cancer and as a therapeutic target in the management of patients with this type of cancer.

A Pilot Study of a Panel of Ocular Inflammation Biomarkers in Patients with Primary Sjögren's Syndrome.

Ocular diseases have a strong impact on individuals, the effects of which extend from milder visual impairment to blindness. Due to this and to their prevalence, these conditions constitute important health, social and economic challenges. Thus, improvements in their early detection and diagnosis will help dampen the impact of these conditions, both on patients and on healthcare systems alike. In this sense, identifying tear biomarkers could establish better non-invasive approaches to diagnose these diseases and to monitor responses to therapy. With this in mind, we developed a solid phase capture assay, based on antibody microarrays, to quantify S100A6, MMP-9 and CST4 in human tear samples, and we used these arrays to study tear samples from healthy controls and patients with Sjögren's Syndrome, at times concomitant with rheumatoid arthritis. Our results point out that the detection of S100A6 in tear samples seems to be positively correlated to rheumatoid arthritis, consistent with the systemic nature of this autoinflammatory pathology. Thus, we provide evidence that antibody microarrays may potentially help diagnose certain pathologies, possibly paving the way for significant improvements in the future care of these patients.

Ssu72 phosphatase is a conserved telomere replication terminator.

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3'-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

Cold-inducible RNA binding protein promotes breast cancer cell malignancy by regulating Cystatin C levels.

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms. We find that CIRBP transcript levels correlate with breast cancer subtype and are an indicator of luminal A/B prognosis. Accordingly, overexpression of CIRBP in nontumoral MCF-10A cells promotes cell growth and clonogenicity, while depletion of CIRBP from luminal A MCF-7 cells has opposite effects. We use RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to identify a set of 204 high confident CIRBP targets in MCF-7 cells. About 10% of these showed complementary changes after CIRBP manipulation in MCF-10A and MCF-7 cells, and were highly interconnected with known breast cancer genes. To test the potential of CIRBP-mediated regulation of these targets in breast cancer development, we focused on Cystatin C (CST3), one of the most highly interconnected genes, encoding a protein that displays tumor suppressive capacities. CST3 depletion restored the effects of CIRBP depletion in MCF-7 cells, indicating that CIRBP functions, at least in part, by down-regulating CST3 levels. Our data provide a resource of CIRBP targets in breast cancer, and identify CST3 as a novel downstream mediator of CIRBP function.

Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis.

Cystatin M/E (encoded by the CST6 gene) is a cysteine protease inhibitor, that exerts regulatory and protective effects against uncontrolled proteolysis mainly by directly regulating cathepsin V, cathepsin L, and legumain activities. Previous studies have suggested that CST6 may exert a regulatory role in epidermal differentiation and hair follicle formation by inhibiting the activity of respective cognate target proteases. However, until recently, studies have revealed that loss- or gain-of-function of the CST6 gene causes dry skin with hypotrichosis in humans. Here, we reported two siblings of Chinese origin with dry skin, desquamation and abnormal keratosis without hypotrichosis. By applying whole-exome sequencing, we identified homozygous loss-of-function mutation c.251G > A (p.Gly84Asp) in the CST6 gene as the underlying genetic cause. Further fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on the protease activity of cathepsins. Moreover, the corresponding mutation in mice resulted in excessive cornification, desquamation, impaired skin barrier function, and abnormal proliferation and differentiation of keratinocytes. In conclusion, the homozygous missense mutation c.251G > A in CST6 gene resulted in dry skin, desquamation, as well as abnormal keratosis of the skin, promoting our understanding of the role of protease-antiprotease balance in human skin disorders.

Epithelium-derived cystatin SN inhibits house dust mite protease activity in allergic asthma.

BACKGROUND: Allergen source-derived proteases are a critical factor in the formation and development of asthma. The cysteine protease activity of house dust mite (HDM) disrupts the epithelial barrier function. The expression of cystatin SN (CST1) is elevated in asthma epithelium. CST1 inhibits the cysteine protease activity. We aimed to elucidate the role of epithelium-derived CST1 in the development of asthma caused by HDM. METHODS: CST1 protein levels in sputum supernatants and serum of patients with asthma and healthy volunteers were measured by ELISA. The ability of CST1 protein to suppress HDM-induced bronchial epithelial barrier function was examined in vitro. The effects of exogenous CST1 protein on abrogating HDM-induced epithelial barrier function and inflammation were examined in mice in vivo. RESULTS: CST1 protein levels were higher in sputum supernatants (142.4 ± 8.95 vs 38.87 ± 6.85 ng/mL, P < 0.0001) and serum (1129 ± 73.82 vs 703.1 ± 57.02 pg/mL, P = 0.0035) in patients with asthma than in healthy subjects. The levels were significantly higher in patients with not well- and very poorly controlled asthma than those with well-controlled asthma. Sputum and serum CST1 protein levels were negatively correlated with lung function in asthma. CST1 protein levels were significantly lower in the serum of HDM-specific IgE (sIgE)-positive asthmatics than in sIgE-negative asthmatics. The HDM-induced epithelial barrier function disruption was suppressed by recombinant human CST1 protein (rhCST1) in vitro and in vivo. CONCLUSION: Our data indicated that human CST1 protein suppresses asthma symptoms by protecting the asthmatic bronchial epithelial barrier through inhibiting allergenic protease activity. CST1 protein may serve as a potential biomarker for asthma control.

CST2 is activated by RUNX1 and promotes pancreatic cancer progression by activating PI3K/AKT pathway.

Cystatin 2 (CST2) is a protein coding gene that belongs to a large superfamily of cysteine protease inhibitors. The deregulation of CST2 has been implicated in human cancers. The role of CST2 in pancreatic carcinogenesis has not yet been investigated. In this study, Gene Expression Profiling Interactive Analysis was performed using the Cancer Genome Atlas (TCGA) dataset containing pancreatic tumor samples and normal tissues. The functional role of CST2 in pancreatic cells was investigated by gene knockdown in vitro and in mouse xenograft tumor model. We found that CST2 was overexpressed in pancreatic tumor samples and cell lines. The knockdown of CST2 led to reduced proliferation, migration, and invasion, while apoptotic events were increased upon CST2 silencing in pancreatic cancer cells. In the xenograft mouse model of pancreatic cells, CST2 knockdown also retarded tumor growth on tumor growth. RUNX1 was identified as a transcription factor which positively regulated the expression of CST2. Further, we showed that, CST2 knockdown suppressed the activation of the PI3K/AKT signaling in pancreatic cells. Overall, our findings suggest that CST2 serves as an oncogene which facilitates the progression of pancreatic cancer. RUNX1 functions to upregulate CST2 in pancreatic cancer cells and CST2 may promote the malignancy of pancreatic cells by maintaining the activation of PI3K/AKT signaling.

Investigating the prognostic and predictive value of the type II cystatin genes in gastric cancer.

BACKGROUND: Accumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated. METHODS: The present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan-Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism. RESULTS: The data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13-3.03, P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS. CONCLUSIONS: Our findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.