The rs1333049 polymorphism on locus 9p21.3 and extreme longevity in Spanish and Japanese cohorts

GeroScience - The rs1333049 (G/C) polymorphism located on chromosome 9p21.3 is a candidate to influence extreme longevity owing to its association with age-related diseases, notably coronary artery...     

The origin and evolution of fragrance in rice (Oryza sativa L.)

Fragrance in the grain is one of the most highly valued grain quality traits in rice, yet the origin and evolution of the betaine aldehyde dehydrogenase gene (BADH2) underlying this trait remains unclear. In this study, we identify eight putatively nonfunctional alleles of the BADH2 gene and show that these alleles have distinct geographic and genetic origins. Despite multiple origins of the fragrance trait, a single allele, badh2.1, is the predominant allele in virtually all fragrant rice varieties today, including the widely recognized Basmati and Jasmine types. Haplotype analysis allowed us to establish a single origin of the badh2.1 allele within the Japonica varietal group and demonstrate the introgression of this allele from Japonica to Indica. Basmati-like accessions were nearly identical to the ancestral Japonica haplotype across a 5.3-Mb region flanking BADH2 regardless of their fragrance phenotype, demonstrating a close evolutionary relationship between Basmati varieties and the Japonica gene pool. These results clarify the relationships among fragrant rice varieties and challenge the traditional assumption that the fragrance trait arose in the Indica varietal group.     

Helicobacter pylori infection,gastrin and cyclooxygenase-2 in gastric carcinogenesis

Gastric cancer is one of the most frequent neoplasms and a main cause of death worldwide, especially in China and Japan. Numerous epidemiological, animal and experimental studies support a positive association between chronic Helicobacter pylori(H. pylori) infec-tion and the development of gastric cancer. However, the exact mechanism whereby H. pylori causes gastric carcinogenesis remains unclear. It has been demon-strated that expression of cyclooxygenase-2(COX-2) is elevated in gastric carcinomas and in their precursor le-sions. In this review, we present the latest clinical and experimental evidence showing the role of gastrin and COX-2 in H. pylori-infected patients and their possible association with gastric cancer risk.     

The Role of CDX2 in Intestinal Metaplasia Evaluated Using Immunohistochemistry

Background/Aims

Intestinal metaplasia (IM) has been regarded as a premalignant condition. This study evaluated the role of the transforming factor CDX2 according to the severity and type of IM.

Methods

This analysis was performed on 383 subjects with IM in the antrum and/or body, with diagnoses that were categorized as controls, dysplasias, and gastric cancers. The IM grades were classified into four groups as negative, mild, moderate or severe using the updated Sydney scoring system. The IM subtypes were categorized as type I, type II, and type III using high iron diamine and alcian blue (pH 2.5) staining. The CDX2 expression in the IM foci was evaluated using immunohistochemistry in specimens from the antrum and/or body.

Results

CDX2 expression increased according to IM severity (p=0.001) but was not associated with the IM subtype (p=0.881) in the antrum specimens. Similarly, CDX2 expression increased according to the IM grade (p=0.001) but was not associated with the IM subtype (p=0.755) in the body specimens. CDX2 expression was also increased according to baseline disease in the antrum, especially dysplastic and GC group (p=0.003), but not in the body (p=0.582). However, status of Helicobacter pylori infection was not associated with CDX2 expression in the antrum (p=0.692) and body (p=0.271).

Conclusions

These results show that CDX2 expression is associated with the IM grade regardless of the IM subtype and that it was more frequent in the dysplasia group. These results suggest that CDX2 expression might play an important role in the progression of IM in various environments that can affect neoplastic change.     

Role for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection

The pathogenic mycobacteria that cause tuberculosis (TB) and TB-like diseases in humans and animals elude sterilizing immunity by residing within an intracellular niche in host macrophages, where they are protected from microbicidal attack. Recent studies have emphasized microbial mechanisms for evasion of host defense; less is known about mycobactericidal mechanisms that remain intact during initial infection. To better understand macrophage mechanisms for restricting mycobacteria growth, we examined Mycobacterium marinum infection of Drosophila S2 cells. Among approximately 1,000 host genes examined by RNAi depletion, the lysosomal enzyme beta-hexosaminidase was identified as an important factor in the control of mycobacterial infection. The importance of beta-hexosaminidase for restricting mycobacterial growth during mammalian infections was confirmed in macrophages from beta-hexosaminidase knockout mice. Beta-hexosaminidase was characterized as a peptidoglycan hydrolase that surprisingly exerts its mycobactericidal effect at the macrophage plasma membrane during mycobacteria-induced secretion of lysosomes. Thus, secretion of lysosomal enzymes is a mycobactericidal mechanism that may have a more general role in host defense.     

KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients

AIM:To investigate gene mutations and DNA mismatch repair(MMR) protein abnormality in Chinese colorectalcarcinoma(CRC) patients and their correlations with clinicopathologic features.METHODS:Clinical and pathological information for 535 patients including 538 tumors was reviewed and recorded.Mutation analyses for exon 2 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing except that in 9 tumors amplification refractory mutation system PCR was used.Expression of MMR proteins including MHL1,MSH2,MSH6 and PMS2 was evaluated by immunohistochemistry.Correlations of KRAS and BRAF mutation status and the expression status of MMR proteins with age,gender,cancer stage,location,and histology were analyzed.Correlations between KRAS or BRAF mutations and MMR protein expression were also explored.RESULTS:The overall frequencies of KRAS and BRAF mutations were 37.9% and 4.4%,respectively.KRAS mutations were more common in patients ≥ 50 years old(39.8% vs 22% in patients 50 years old,P 0.05).The frequencies of BRAF mutants were higher in tumors from females(6.6% vs males 2.8%,P 0.05),located in the right colon(9.6% vs 2.1% in the left colon,1.8% in the rectum,P 0.01),with mucinous differentiation(9.8% vs 2.8% without mucinous differentiation,P 0.01),or being poorly differentiated(9.5% vs 3.4% well/moderately differentiated,P 0.05).MMR deficiency was strongly associated with proximal location(20.5% in the right colon vs 9.2% in the left colon and 5.1% in the rectum,P 0.001),early cancer stage(15.0% in stages Ⅰ-Ⅱ vs 7.7% in stages Ⅲ-Ⅳ,P 0.05),and mucinous differentiation(20.2% vs 9.2% without mucin,P 0.01).A higher frequency of MLH1/PMS2 loss was found in females(9.2% vs 4.4% in males,P 0.05),and MSH2/MSH6 loss tended to be seen in younger(50 years old) patients(12.0% vs 4.0% ≥ 50 years old,P 0.05).MMR deficient tumors were less likely to have KRAS mutations(18.8% vs 41.7% in MMR proficient tumors,P 0.05) and tumorswith abnormal MLH1/PMS2 tended to harbor BRAF mutations(15.4% vs 4.2% in MMR proficient tumors,P 0.05).CONCLUSION:The frequency of sporadic CRCs having BRAF mutation,MLH1 deficiency and MSI in Chinese population may be lower than that in the Western population.     

Functional interaction between native G protein-coupled purinergic receptors in Xenopus follicles

Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K⁺ current (I_K,cAMP), whereas ATP activates a Cl⁻ current (F_Cl) and has a dual effect on I_K,cAMP, provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (i_etr/i_wf) in the two preparations. For ATP activation of I_K,cAMP and F_Cl, the ratios i_etr/i_wf were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (I_K,cAMP) was 14 ± 3.8 μM [Hill coefficient (nH) = 2.7 ± 0.61], and that in e.t.r. follicles (F_Cl) was 1.8 ± 0.68 μM (nH = 0.76 ± 0.09), whereas Ado-response parameters did not change. Responses to UTP and β,γ-methylene-ATP, specific agonists for I_K,cAMP inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.     

TET2 mutations in secondary acute myeloid leukemias: a French retrospective study

Ten-eleven translocation 2 (TET2) mutations have been involved in myeloid malignancies. This retrospective study aims at evaluating the frequency and impact of TET2 mutations in 247 secondary acute myeloid leukemia cases referred to as myelodysplasia-related changes (n=201) or therapy-related (n=46) leukemias. Mutation of at least one copy of the TET2 gene was detected in 49 of 247 (19.8%) patients who presented with older age, higher hemoglobin level, higher neutrophil and monocyte counts, and lower platelet count. TET2 mutations were significantly less frequent in therapy-related (8.7%) than myelodysplasia-related changes (22.3%; P=0.035) leukemias and strongly associated with normal karyotype (P<0.001). TET2 mutations did not significantly associate with NPM1, FLT3-ITD or FLT3-D835, WT1, or N- or K-RAS mutations. Complete remission was achieved in 57% of evaluable patients who had received intensive chemotherapy. In this group, TET2 mutations did not influence the complete remission rate or overall survival.     

The COX-2-1195AA Genotype Is Associated with Diffuse-Type Gastric Cancer in Korea

Background/Aims

The potential role of the cyclooxygenase (COX)-2 polymorphism has been reported in relation to the risk of gastrointestinal tract malignancies. Therefore, we investigated whether COX-2 polymorphisms are associated with the risk of gastric cancer (GC) in Korea, one of the areas with a high prevalence of this condition.

Methods

We evaluated the genotypic frequencies of COX-2-765 and -1195 in 100 peptic ulcer patients, 100 GC patients, and 100 healthy controls. The polymorphisms of the COX-2-765 and -1195 genes were analyzed by polymerase chain reaction and restriction fragment length polymorphisms.

Results

The frequencies of the COX-2-1195 GG, GA, and AA genotype were 20%, 60%, and 20% in intestinal-type GC and 8%, 48%, and 44% in diffuse-type GC, respectively (p=0.021). There were no significant differences in the frequency of COX-2-765 genotypes between intestinal-type GC and diffuse-type GC (p=0.603). Age- and sex-adjusted logistic regression analysis showed that the COX-2-1195 AA genotype was the independent risk factor of diffuse-type GC compared with the COX-2-1195 GG genotype (p=0.041; odds ratio, 6.22; 95% confidence interval, 1.077 to 35.870).

Conclusions

The COX-2-1195 AA genotype may render subjects more susceptible to diffuse-type GC.     

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as “stitchers,” to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication–licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.Transitional cell carcinoma arising in the urinary bladder (TCC-UB) is a frequent cause of morbidity and mortality, and among patients in the United States, it is one of the most costly cancers to treat (1, 2). The traditional somatic genetic basis of TCC-UB is a distinct division of low-grade papillary tumors from high-grade invasive tumors. Low-grade papillary superficial tumors are generally characterized by constitutive activation of the receptor tyrosine kinase–Ras pathway, and they have activating mutations in the HRAS and fibroblast growth factor receptor 3 (FGFR3) genes (3–6). In contrast, high-grade invasive TCC-UB is characterized by alterations in the tumor protein p53 (TP53) and retinoblastoma 1 (RB1) pathways. These genes normally regulate the cell cycle by interacting with the Ras–mitogen-activated protein kinase signal transduction pathway (7, 8). Both low-grade papillary and high-grade invasive tumors frequently have loss of chromosome 9. This loss presumably inactivates the p16 gene and is an early event in the initiation of TCC-UB (9, 10)Although TP53, cyclin-dependent kinase inhibitor 2A (p16), RB1, HRAS, and FGFR3 abnormalities have been well described in TCC-UB, there are limited data on the more complete genomic analysis of TCC-UB (11). A recent study focusing on genome-wide copy number analysis showed extensive heterogeneity across all subtypes of TCC-UB to such an extent that precise molecular groupings were difficult to define (12). In this study, similar to earlier studies in melanoma (13) and medulloblastoma (14), evidence of the association of TP53 mutations with specific copy number alterations, referred to as chromothripsis, was noted. The study in medulloblastoma (14) was particularly intriguing in that identification of a molecular subclass with TP53 mutations was associated with chromothripsis and a more aggressive clinical outcome was noted. Chromothripsis, or the shattering of two or more chromosomes and their reassembly into derivative chromosomes, is different from other types of genomic instability, which tend to occur on a genome-wide basis (15, 16). Chromothripsis is different in that it includes one to three alternating copy number states across the derivative chromosome, an association with changes in heterozygosity, and numerous genomic rearrangements in localized chromosomal regions likely occurring in condensed chromosomes (15). There is evidence to suggest that the primary mechanism of reassembly of the derivative chromosome in chromothripsis is nonhomologous end-joining (NHEJ) (14). With the advent of next-generation sequencing (NGS) allowing for detailed genomic analysis, chromothripsis has been identified more frequently (17). Additionally, the presence of these complex genomic events and their potential association with TP53 mutations may contribute to a better understanding of cancer, including TCC-UB.NGS technologies provide other evidence of complex genomic heterogeneity, such as the recent identification of subclonal populations of cells with mutations distinct from the dominant clonal population of cells within one tumor or between a primary, recurrent, or metastatic tumor from one patient (18–20). Importantly, a recent study in chronic lymphocytic leukemia (CLL) (21) showed how selective pressures on cancer cells, such as chemotherapy, select for these subclonal populations to become the dominant clone contributing to genomic heterogeneity. It is not yet certain whether broad measurements of genomic heterogeneity will have an impact on molecular classification of cancer, but it is likely that they will significantly contribute to biological differences, and therefore have an impact on patient outcomes.To evaluate the spectrum of genomic heterogeneity in TCC-UB, we performed complete genome sequencing of five high-grade muscle-invasive tumors and matching germ-line blood (SI Appendix, Table S1), and validated a subset of our findings in more than 300 bladder cancer specimens. Our overall results showed a great deal of genomic heterogeneity at either extreme of a spectrum of genomic complexity.     

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical–basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.Organ growth is determined by cell numbers produced by meristems and by cell expansion to reach final volume. Plant hormones steer the extent and timing of growth and mediate signals of various types that are transmitted within the cell, between cells, or at a long distance within the plant. The phytohormone auxin is a major regulator of cell division and expansion during plant growth and development. The molecular mechanisms by which auxin controls these essential cellular responses are roughly understood thanks to the recent progress in the identification of auxin receptors and components of auxin signaling, transport, and metabolism (1). Auxin gradients between the cells are generated and maintained by intercellular auxin transport mediated by efflux carriers from the PIN-FORMED (PIN) family (2). PIN proteins contain transmembrane domains and continuously cycle between the basal (rootward) and apical (shootward) plasma membranes and endosomes, allowing rapid and dynamic changes in the PIN localization (3). The sorting of PIN proteins into the apical or basal trafficking pathway depends on the PIN phosphorylation status, which is controlled by the PINOID (PID) protein kinase and phosphatase 2A (PP2A) (4, 5), a heterotrimeric complex consisting of a C-catalytic subunit together with A- and B-regulatory subunits. One of the A-subunit isoforms, ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), acts as a key positive regulator of the PP2A activity in seedlings. The rcn1 mutant that lost part of the PP2A activity displays abnormalities related to defective auxin transport, such as altered gravity response and lateral root growth (6, 7).In an ethyl methanesulfonate-induced collection of Arabidopsis thaliana leaf mutants (8), we identified ROTUNDA3 (RON3) as a proline-rich, plant-specific single-copy gene with a function in auxin-related processes in all organs throughout the plant’s lifecycle. Affinity purification of the PP2A complex with RON3 as bait, and genetic and cell biology analyses support the hypothesis that RON3 affects the cellular dynamics of PIN proteins through interference with the PP2A activity.     

Differential expression of pancreatic protein and chemosensing receptor m RNAs in NKCC1-null intestine

AIM: To investigate the intestinal functions of the NKCC1 Na~+-K~+-2Cl cotransporter(SLC12a2 gene), differential m RNA expression changes in NKCC1-null intestine were analyzed.METHODS: Microarray analysis of m RNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice(n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed.RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas m RNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.     

The H19 locus acts in vivo as a tumor suppressor

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms' tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.     

Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells

Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle–nucleus replicational coupling is an evolutionary conserved process among plant cells.     

Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations ("two-hit" cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal-epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.