Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

FMR1 allelic complexity in premutation carriers provides no evidence for a correlation with age at amenorrhea.

BACKGROUND: Premutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, defined as between 55 and 200 CGGs, have been implicated in fragile X-associated primary ovarian insufficiency (FXPOI). Only 20% of female premutation carriers develop early ovulatory dysfunction, the reason for this incomplete penetrance is unknown. This study validated the mathematical model in premutation alleles, after assigning each allele a score representing allelic complexity. Subsequently, allelic scores were used to investigate the impact of allele complexity on age at amenorrhea for 58 premutation cases (116 alleles) previously published. METHODS: The allelic score was determined using a formula previously described by our group. The impact of each allelic score on age at amenorrhea was analyzed using Pearson's test and a contour plot generated to visualize the effect. RESULTS: Correlation of allelic score revealed two distinct complexity behaviors in premutation alleles. No significant correlation was observed between the allelic score of premutation alleles and age at amenorrhea. The same lack of significant correlation was observed regarding normal-sized alleles, despite a nearly significant trend. CONCLUSIONS: Our results suggest that the use of allelic scores combination have the potential to explain female infertility, namely the development of FXPOI, or ovarian dysfunction, despite the lack of correlation with age at amenorrhea. Such a finding is of great clinical significance for early identification of females at risk of ovulatory dysfunction, enhancement of fertility preservation techniques, and increasing the probability for a successful pregnancy in females with premutations. Additional investigation is necessary to validate this hypothesis.

Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes.

We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz-Kaveggia syndrome, Lujan-Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype-phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations.

What Are the Effects on Palate of Early Lip Surgery in Children With Cleft Lip and Palate? Cross-Sectional Evaluation From 5-Year-Old.

This study aimed to evaluate the postsurgical effects from 5 years on the palate after surgical repair of the lip at 3 or 9 months of age in children with cleft lip and palate. Eighty-four digitized dental impressions were divided into the following groups: group 1 (G1): lip surgery at 3 months of life; group 2 (G2): lip surgery at 9 months of life; group 3 (G3): without orofacial cleft. Five angular (C'IC, ICM, IC'M', CMM', and C'M'M) and 3 linear parameters (C-C', c-c', and M-M') were evaluated. Statistical analysis was applied with α=5%. Intraclass Correlation Coefficient was significantly smaller in G1 than in G3 ( P =0.005), while IC'M' was significantly smaller in G3 than in G1 ( P <0.001). C'M'M was significantly smaller in G1 than in G2 and G3 ( P <0.001). The distances C-C' and c-c' were significantly smaller in G1 than in G2 and G3 ( P <0.001). There was a statistically significant difference in both G1 and G2 ( P <0.001, in all) in the analysis of palatal symmetry. Linear regression analysis showed that the, 11.2% of outcomes determined by c-c' distance can be explained by the age of lip repair ( P =0.013). In conclusion, lip surgery at 3 months of life showed a tendency toward more restriction in 5-year postsurgery palate development. The age of cheiloplasty is one of the factors that can influence palatal development; however, other factors may be associated and should be studied.

Customized Tray for Impression Taking in Children With Cleft Lip and Palate.

The assessment of rehabilitation outcomes requires a patient documentation protocol, including records obtained at standardized ages, to compare different types of surgeries, their effects, as well as between different rehabilitation centers. The aim of this paper was to present proper trays for babies with different types of cleft lip and palate, which are used in the outpatient routine at Hospital of Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP). The customized trays are made with self-curing acrylic resin. The tray must have suitable depth to copy the buccal sulcus, and wax is usually applied to contour the tray edge, and the adjustment of the tray to the fornix, making the tray specific for each child. The impression precludes the utilization of dental casts for diagnosis, treatment plan, and research measurements. In the clinical practice at HRAC-USP, it was observed that customized trays increased the quality of impression, accurately reproducing anatomical features of dental arches of babies with oral clefts.

3D morphometric evaluation of the dental arches in children with cleft lip and palate submitted to different surgical techniques.

OBJECTIVES: This study aimed to compare dimensional alterations of dental arches in children with unilateral complete cleft lip and palate before and after different techniques of primary plastic surgeries. MATERIALS AND METHODS: The sample was divided into two groups: group 1-cheiloplasty by Millard's technique and one-stage palatoplasty by von Langenbeck's technique; group 2-cheiloplasty by Millard's technique and two-stage palatoplasty: anterior palatoplasty by Hans Pichler's technique and posterior palatoplasty by Sommerlad's technique. Dental arches were evaluated before (T1), after the first phase (T2), and 1 year after the second phase (T3) of primary surgeries. Linear measurements and palatal area were assessed. To analyze the method's error, interclass correlation coefficient was applied. ANOVA (followed by Tukey test), dependent, and independent t-test were used (p < 0.05). RESULTS: At T1, the intertuberosity distance was statistically greater in G2 (p = 0.004). At T2, the anterior length of the dental arch was statistically greater in G2 (p = 0.025), while the area of the smaller palatal segment (p = 0.001), cleft area (p = 0.014), and total area (p = 0.002) were statistically smaller in G2. At T3, the intertuberosity distance was statistically greater in G2 (p = 0.017). CONCLUSION: This study suggests that cheiloplasty and one-stage palatoplasty resulted in smaller growth of maxilla than cheiloplasty and two-stage palatoplasty in the linear measurements (T-T' and I-CC') and total area of the dental arches. CLINICAL RELEVANCE: Surgical protocols need to be evaluated to verify their effects aiming at improving the clinical practice of the interdisciplinary team, determining new parameters for the rehabilitation of individuals with cleft lip and palate.

Intellectual disability genomics: current state, pitfalls and future challenges.

Intellectual disability (ID) can be caused by non-genetic and genetic factors, the latter being responsible for more than 1700 ID-related disorders. The broad ID phenotypic and genetic heterogeneity, as well as the difficulty in the establishment of the inheritance pattern, often result in a delay in the diagnosis. It has become apparent that massive parallel sequencing can overcome these difficulties. In this review we address: (i) ID genetic aetiology, (ii) clinical/medical settings testing, (iii) massive parallel sequencing, (iv) variant filtering and prioritization, (v) variant classification guidelines and functional studies, and (vi) ID diagnostic yield. Furthermore, the need for a constant update of the methodologies and functional tests, is essential. Thus, international collaborations, to gather expertise, data and resources through multidisciplinary contributions, are fundamental to keep track of the fast progress in ID gene discovery.

Discerning the role of polymicrobial biofilms in the ascent, prevalence, and extent of heteroresistance in clinical practice.

Antimicrobial therapy is facing a worrisome and underappreciated challenge, the phenomenon of heteroresistance (HR). HR has been gradually documented in clinically relevant pathogens (e.g. Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia spp., Acinetobacter baumannii, Klebsiella pneumoniae, Candida spp.) towards several drugs and is believed to complicate the clinical picture of chronic infections. This type of infections are typically mediated by polymicrobial biofilms, wherein microorganisms inherently display a wide range of physiological states, distinct metabolic pathways, diverging refractory levels of stress responses, and a complex network of chemical signals exchange. This review aims to provide an overview on the relevance, prevalence, and implications of HR in clinical settings. Firstly, related terminologies (e.g. resistance, tolerance, persistence), sometimes misunderstood and overlapped, were clarified. Factors generating misleading HR definitions were also uncovered. Secondly, the recent HR incidences reported in clinically relevant pathogens towards different antimicrobials were annotated. The potential mechanisms underlying such occurrences were further elucidated. Finally, the link between HR and biofilms was discussed. The focus was to recognize the presence of heterogeneous levels of resistance within most biofilms, as well as the relevance of polymicrobial biofilms in chronic infectious diseases and their role in resistance spreading. These topics were subject of a critical appraisal, gaining insights into the ascending clinical implications of HR in antimicrobial resistance spreading, which could ultimately help designing effective therapeutic options.

A Biobank of Stem Cells of Human Exfoliated Deciduous Teeth: Overview of Applications and Developments in Brazil.

A biobank is an organized collection of biological human material and its associated information stored for research according to regulations under institutional responsibility, without commercial purposes, being a mandatory and strategical activity for research, regenerative medicine, and innovation. Stem cells have largely been employed in research and frequently stored in biobanks, which have been used as an essential source of biological materials. Stem cells of human exfoliated deciduous teeth (SHED) are stem cells which have a high multipotency and can be easily obtained. Besides, this extremely accessible tissue has advantages with respect to storage, as the SHED obtained in childhood can be used in later life, which implies the necessity for the creation and regulation of biobanks. The proper planning for the creation of a biobank includes knowledge of the material types to be stored, requirements regarding handling and storage conditions, storage time, and room for the number of samples. Thus, this study aimed to establish an overview of the development of a SHED biobank. Ethical and legal standardization, current applications, specific orientations, and challenges for the implementation of a SHED biobank were discussed. Through this overview, we hope to encourage further studies to use SHED biobanks.

Salivary proteomic profile of dogs with and without dental calculus.

BACKGROUND: Dogs' saliva is a complex mixture of inorganic and organic constituents, rich in proteins. Therefore, knowing the saliva composition of these animals is extremely important to identify the presence of proteins that may be involved in physiological and pathological mechanisms of their oral cavity. The present study aimed to characterize the proteomic profile of saliva from dogs with and without dental calculus. RESULTS: Saliva samples were collected from 20 dogs. Before the collection, a visual clinical examination was performed and 8 subjects (40%) did not present any signs of dental calculus, while 12 (60%) presented dental calculus. After saliva collection, the samples were submitted to protein quantification (mBCA), and then they were prepared for analysis by nLC-ESI-MS/MS. A total of 658 unique proteins were identified, of which 225 were specific to dogs without dental calculus, 300 were specific to dogs with dental calculus, and 133 were common to all subjects. These proteins presented functions including transportation, immune response, structural, enzymatic regulation, signal transduction, transcription, metabolism, and some proteins perform functions as yet unknown. Several salivary proteins in dogs with dental calculus differed from those found in the group without dental calculus. Among the abundant proteins detected in periodontal affected cases, can be highlighting calcium-sensing receptor and transforming growth factor beta. Enrichment analysis reveled the presence of Rho GTPases signaling pathway. CONCLUSIONS: This research identified salivary proteins, that should be further investigated as potencial biomarkers of chronic periodontits with dental calculus formation in dogs.

Pseudomonas aeruginosa and Staphylococcus aureus communication in biofilm infections: insights through network and database construction.

The polymicrobial nature of most infections is often characterized by complex biofilm communities, where pathogen interactions promote infection progression and severity. Quorum-sensing, the major regulator of virulence and inter-species communication, is a promising target for new anti-infective strategies. This study aimed at collecting and analysing experimental information on the molecular basis of Pseudomonas aeruginosa and Staphylococcus aureus interactions in biofilms. Data were systematically annotated from relevant full-text papers optimally retrieved from PubMed, reconstructed as networks and integrated with specialized databases to identify promising antimicrobial targets. Network analysis revealed key entities regulating P. aeruginosa/S. aureus interactions, for instance the PqsABCDE/PqsR quorum-sensing system, which affects S. aureus growth and biofilm formation. By identifying the most reported P. aeruginosa virulence factors affecting S. aureus, for example, HQNO and siderophores, a list of experimentally validated agents affecting those factors, ranging from synthetic drugs to natural plant extracts, was constructed. The complex experimental data on P. aeruginosa/S. aureus interactions were for the first time systematically organized and made publically available in the new Inter-Species CrossTalk Database (www.ceb.uminho.pt/ISCTD).

Analysis of Dental Arch in Children With Oral Cleft Before and After the Primary Surgeries.

This study aimed to evaluate longitudinally the alteration of the dental arch dimensions of children with different oral cleft types, before and after the primary surgeries. Three-dimensional images of the maxillary dental casts of children with unilateral complete cleft lip (G1), unilateral complete cleft lip and palate (G2), and cleft palate (G3). The children were evaluated at pre-cheiloplasty (T1), pre-palatoplasty (T2), and 1 year after palatoplasty (T3). The measurements obtained: intercanine (C-C') and intertuberosity (T-T') distances, anterior (I-CC') and total (I-TT') arch lengths. To analyze the intraexaminer error, paired t-test was applied and Dahlbergh formula. The intragroups comparisons were applied: paired t-test, ANOVA followed by Tukey, Wilcoxon test, and Kruskal-Wallis test followed by Dunn test. The intergroup comparisons were performed by independent t-test and Mann-Whitney test. In G1, the C-C', T-T', and the I-TT' distances revealed a significant increase of the maxilla. In G2, the C-C' distance statistically decreased from T1 to T3, T-T' distance showed statistical increase from T1 to T3. The I-TT' length increased with statistically significant differences between T1 and T3, T2 and T3. In G3, the C-C', T-T', and I-TT' distance increased was statistically significant. The longitudinal evaluation of the changes occurred in the dental arches with different oral cleft types showed that cheiloplasty and palatoplasty caused the most alterations in the development of the maxillary dimensions of children with complete cleft lip and palate.

Classical fragile-X phenotype in a female infant disclosed by comprehensive genomic studies.

BACKGROUND: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. CASE PRESENTATION: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. CONCLUSIONS: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.

Exploring anti-quorum sensing and anti-virulence based strategies to fight Candida albicans infections: an in silico approach.

The complex virulence attributes of Candida albicans are an attractive target to exploit in the development of new antifungals and anti-virulence strategies to combat C. albicans infections. Particularly, quorum sensing (QS) has been reported as critical for virulence regulation in C. albicans. This work presents two knowledge networks with up-to-date information about QS regulation and experimentally tested anti-QS and anti-virulence agents for C. albicans. A semi-automatic bioinformatics workflow that combines literature mining and expert curation was used to retrieve otherwise scattered information from the scientific literature. The network representation offers an innovative and continuously updatable means for the Candida research community to query QS and virulence data systematically and in a user-friendly way. Notably, the reconstructed networks show the complexity of QS regulation and the impact that some molecules have on the inhibition of virulence mechanisms responsible for infection establishment (e.g. hyphal development) and perseverance (e.g. biofilm formation). In the future, the compiled knowledge may be used to build decision-making models that help infer new knowledge of practical significance. The knowledge networks are publicly available at http://pcquorum.org/. This Web platform enables the exploration of fungal virulence cues as well as reported inhibitors in a user-friendly fashion.

Anthropometric Analysis of the Dental Arches of Five-Year-Old Children With Cleft Lip and Palate.

This study aimed to analyze the dimensional alterations of the dental arches of 5-year-old children with cleft lip and palate and to compare these dimensions with children without oral clefts. One hundred twenty children were divided into the following groups: unilateral cleft lip (UCL), unilateral cleft lip and palate (UCLP), cleft palate (CP), and control (C). A specific software was used to digitize the dental casts and perform the anthropometric analyses through the measurement of transversal linear intercanine and intermolar distances on the maxilla and mandible. The intergroup comparisons of the maxillary dimensions exhibited that the intercanine distances of groups C and UCL were statistically greater than that of groups UCLP and CP. The intermolar distance was significantly smaller in group UCLP than in the other groups. No statistically significant difference occurred in the mandibular intercanine and intermolar distance among groups. The analysis of the superposition of the maxillary over the mandibular transversal distances showed statistically significant differences among groups. This study showed that at 5-year old, the children with cleft involving the palate had more maxillary dimensional alterations than those without cleft palate.

Contraction of fully expanded FMR1 alleles to the normal range: predisposing haplotype or rare events?

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is due to the expansion over 200 CGGs and methylation of this polymorphic region, in the 5'-UTR (untranslated region) of FMR1 (Xq27.3). We have identified four FXS mosaic males: M1-(CGG)35/(CGG)>200; M2-(CGG)26/(CGG)>200; M3-(CGG)39/(CGG)>200; and M4-(CGG)18/(CGG)125/(CGG)>200. After genotyping their respective mothers, we suggested that normal alleles of these patients resulted from post-zygotic contractions of full expansions. The detection of these four rare independent cases led us to hypothesize the existence of a large-contraction predisposing haplotype in our population. Next, we questioned whether other normal pure CGGs would have arisen through similar contractions from fully expanded alleles. To address these questions, we identified stable single-nucleotide polymorphism (SNP) lineages and related short tandem repeat (STR) haplotypes (DXS998-DXS548-FRAXAC1-FRAXAC2) of the four mosaics, 123 unrelated FXS patients and 212 controls. An extended flanking haplotype (34-44-38-336) shared by mosaics from lineage A suggested a risk lineage-specific haplotype more prone to large contractions. Other normal pure FMR1 alleles from this SNP background also shared phylogenetically close STR haplotypes, although a single (CGG)exp>(CGG)24 contraction or the loss of AGG interruptions may explain their origin. In both scenarios, multistep FMR1 mutations involving the gain or loss of several CGGs seem to underlie the evolution of the repeat.

Quorum sensing inhibition in Pseudomonas aeruginosa biofilms: new insights through network mining.

Quorum sensing plays a pivotal role in Pseudomonas aeruginosa's virulence. This paper reviews experimental results on antimicrobial strategies based on quorum sensing inhibition and discusses current targets in the regulatory network that determines P. aeruginosa biofilm formation and virulence. A bioinformatics framework combining literature mining with information from biomedical ontologies and curated databases was used to create a knowledge network of potential anti-quorum sensing agents for P. aeruginosa. A total of 110 scientific articles, corresponding to 1,004 annotations, were so far included in the network and are analysed in this work. Information on the most studied agents, QS targets and methods is detailed. This knowledge network offers a unique view of existing strategies for quorum sensing inhibition and their main regulatory targets and may be used to readily access otherwise scattered information and to help generate new testable hypotheses. This knowledge network is publicly available at http://pcquorum.org/ .

Data Quality in Biofilm High-Throughput Routine Analysis: Intralaboratory Protocol Adaptation and Experiment Reproducibility.

Biofilm research is growing more diverse and dependent on high-throughput technologies, and the large-scale production of results aggravates data substantiation. In particular, experimental protocols are often adapted to meet the needs of a particular laboratory, and no statistical validation of the modified method is provided. This paper discusses the impact of intralaboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non- rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intralaboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.

Challenges in classifying human chromosomal heteromorphisms using banding cytogenetics: From controversial guidelines to the need for a universal scoring system.

Chromosomal heteromorphisms (CHs) are morphological variations predominantly found in constitutive heterochromatic regions of the genome, primarily composed of tandemly repetitive sequences of satellite DNA. Although not completely devoid of genes, these regions are typically not transcribed into proteins and lack obvious phenotypic impact. Nonetheless, their clinical importance is increasingly under scrutiny, with several studies aiming to assess their influence on human diseases and susceptibilities, especially as they are seemingly part of the long noncoding RNAs in certain tissues. This article summarizes the classification methods of human heterochromatic CHs documented in the literature over the last two decades. Multiple scoring systems have been identified, and previous approaches for CH assessment and reporting in genetic diagnosis have shown inconsistencies. Owing to the current heterogeneity in the classification of CHs, data analysis may be biased, impacting the quality of clinical reports and human genetic research. This review highlights the need for a universal scoring system, which is essential for scientific reproducibility and the accurate identification and clinical evaluation of human CHs.