Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Adapting to children's individual language proficiency: An observational study of preschool teacher talk addressing monolinguals and children learning English as an additional language.

In an increasingly diverse society, young children are likely to speak different first languages that are not the majority language of society. Preschool might be one of the first and few environments where they experience the majority language. The present study investigated how preschool teachers communicate with monolingual English preschoolers and preschoolers learning English as an additional language (EAL). We recorded and transcribed four hours of naturalistic preschool classroom activities and observed whether and how preschool teachers tailored their speech to children of different language proficiency levels and linguistic backgrounds (monolingual English: n = 13; EAL: n = 10), using a suite of tools for analysing quantity and quality of speech. We found that teachers used more diverse vocabulary and more complex syntax with the monolingual children and children who were more proficient in English, showing sensitivity to individual children's language capabilities and adapting their language use accordingly.

Incidence and clinical characteristics of pediatric-onset type 2 diabetes in Hong Kong: The Hong Kong childhood diabetes registry 2008 to 2017.

OBJECTIVE: With increasing prevalence of childhood obesity worldwide, the incidence of pediatric-onset type 2 diabetes (T2D) is also increasing in many countries. We aim to analyze the time trend and incidence of T2D in children in Hong Kong from 2008 to 2017, and to characterize clinical characteristics at diagnosis. METHODS: Data were retrieved from the Hong Kong Childhood Diabetes Registry. All children with T2D diagnosed at the age of less than 18 years from January 1, 2008 to December 31, 2017 and managed in the public health care system were included in this study. RESULTS: In the incident years of 2008-2017 period, 391 children were diagnosed with T2D. The crude incidence rate was 3.42 per 100,000 persons/year [95% confidence interval (CI) 3.08-3.76], which was much higher than that in last registry of 1.27 per 100,000 persons/year in 1997-2007 (P < 0.001).Most children (76%) were asymptomatic and were diagnosed by routine screening. At presentation, a significant proportion presented with co-morbidities including fatty liver (37.9%), dyslipidaemia (35.3%), hypertension (22.5%), and microalbuminuria (12.8%). CONCLUSIONS: The incidence of T2D in children has increased significantly in Hong Kong. Most of them were asymptomatic and picked up on routine health screening. Yet, comorbidities were commonly identified at diagnosis.

Increasing incidence of type 1 diabetes among Hong Kong children and adolescents: The Hong Kong Childhood Diabetes Registry 2008 to 2017.

OBJECTIVE: The incidence of childhood-onset type 1 diabetes (T1D) has been reported to be rising but there is also evidence that it has been attenuated in recent years. We described the time trends and the incidence of T1D in children in Hong Kong from 2008 to 2017 and compared with the previous local registry in 1997 to 2007. METHODS: Data were extracted from the Hong Kong Childhood Diabetes Registry, which was established in 2016. It consists of a retrospective registry (including all childhood diabetes diagnosed in 2008 to 2015) and a prospective registry (including all T1D children diagnosed from 2016 onwards). All T1D children diagnosed at the age of less than 18 years from 1 January 2008 to 31 December 2017 and managed in the public system were included in this study. RESULTS: For the incident years in the 2008 to 2017 period, a total of 498 children with T1D was identified. The crude incidence rate was 4.3 per 100 000 person/year (95% confidence interval 3.96-4.72), which was much higher than the last registry of 2.2 per 100 000 persons/year. Using general linear model, the increment is statistically significant (P = .02). When compared to the last registry, the rate of increment had attenuated, with annual increment in crude incidence in the two periods for T1D <15 years changing from 4.3% to 3.5% (P = .02). CONCLUSIONS: The incidence of T1D children increased significantly in the past two decades in Hong Kong, but the rate of increase had attenuated in recent years.

Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.

Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor-mediated upregulation of caveolin-1.

Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor-dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.

Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-ß and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.

A unified approach to demographic data collection for research with young children across diverse cultures.

Culture is a key determinant of children's development both in its own right and as a measure of generalizability of developmental phenomena. Studying the role of culture in development requires information about participants' demographic backgrounds. However, both reporting and treatment of demographic data are limited and inconsistent in child development research. A barrier to reporting demographic data in a consistent fashion is that no standardized tool currently exists to collect these data. Variation in cultural expectations, family structures, and life circumstances across communities make the creation of a unifying instrument challenging. Here, we present a framework to standardize demographic reporting for early child development (birth to 3 years of age), focusing on six core sociodemographic construct categories: biological information, gestational status, health status, community of descent, caregiving environment, and socioeconomic status. For each category, we discuss potential constructs and measurement items and provide guidance for their use and adaptation to diverse contexts. These items are stored in an open repository of context-adapted questionnaires that provide a consistent approach to obtaining and reporting demographic information so that these data can be archived and shared in a more standardized format. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Heme metabolism and erythropoiesis.

PURPOSE OF REVIEW: Heme biosynthesis requires a series of enzymatic reactions that take place in the cytosol and the mitochondria as well as the proper intercellular and intracellular trafficking of iron. Heme can also be acquired by intestinal absorption and intercellular transport. The purpose of this review is to highlight recent work on heme and iron transport with an emphasis on their relevance in erythropoiesis. RECENT FINDINGS: Whereas the enzymes responsible for heme biosynthesis have been identified, transport mechanisms for iron, heme, or heme synthesis intermediates are only emerging. Recent studies have shed light on how these molecules are transported among various cellular compartments, as well as tissues. Much of this progress can be attributed to the use of model organisms such as S. cerevisiae, C. elegans, D. rerio, and M. musculus. Genetic studies in these models have led to the identification of several new genes involved in heme metabolism. Although our understanding has greatly improved, it is highly likely that other regulators exist and additional work is required to characterize the pathways by which heme and iron are transported within the erythron. SUMMARY: The identification of heme and iron transport mechanisms will improve our understanding of blood development and provide new insight into human blood disorders.

The sound of silence: Reconsidering infants' object categorization in silence, with labels, and with nonlinguistic sounds.

A large body of research based on a specific stimulus set (dinosaur/fish) has argued that auditory labels and novel communicative signals (such as beeps used in a communicative context) facilitate category formation in infants, that such effects can be attributed to the auditory signals' communicative nature, and that other auditory stimuli have no effect on categorization. A contrasting view, the auditory overshadowing hypothesis, maintains that auditory signals disrupt processing of visual information and, therefore, interfere with categorization, with more unfamiliar sounds having a more disruptive effect than familiar ones. Here, we used the dinosaur/fish stimulus set to test these contrasting theories in two experiments. In Experiment 1 (N = 17), we found that 6-month-old infants were able to form categories of these stimuli in silence, weakening the claim that labels facilitated their categorization in infants. These results imply that prior findings of no categorization of these stimuli in the presence of nonlinguistic sounds must be due to disruptive effects of such sounds. In Experiment 2 (N = 17), we showed that familiarity modulated the disruptive effect of nonlinguistic sounds on infants' categorization of these stimuli. Together, these results support the auditory overshadowing hypothesis and provide new insights into the interaction between visual and auditory information in infants' category formation.

Special AT-rich binding protein-2 (SATB2) differentially affects disease-causing p63 mutant proteins.

p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins.

SATB2 augments ΔNp63α in head and neck squamous cell carcinoma.

ΔNp63α is a critical pro-survival protein overexpressed in 80% of head and neck squamous cell carcinomas (HNSCCs) where it inhibits TAp73ß transcription of p53-family target genes, which is thought to increase HNSCC resistance to chemotherapy-induced cell death. However, the mechanisms governing ΔNp63α function are largely unknown. In this study, we identify special AT-rich-binding protein 2 (SATB2) as a new ΔNp63α-binding protein that is preferentially expressed in advanced-stage primary HNSCC and show that SATB2 promotes chemoresistance by enhancing ΔNp63α-mediated transrepression by augmenting ΔNp63α engagement to p53-family responsive elements. Furthermore, SATB2 expression positively correlates with HNSCC chemoresistance, and RNA interference-mediated knockdown of endogenous SATB2 re-sensitizes HNSCC cells to chemotherapy- and γ-irradiation-induced apoptosis, irrespective of p53 status. These findings unveil SATB2 as a pivotal modulator of ΔNp63α that governs HNSCC cell survival.

Predictors of clinical deterioration during hospitalization following acute ischemic stroke.

BACKGROUND/AIMS: A number of risk factors for early worsening of neurological symptoms have been identified. We aimed to evaluate the influence of hemorheologic, biochemical, and metabolic factors on neurological deterioration during hospitalization following acute ischemic stroke and develop a model of neurological deterioration. METHODS: Worsening of stroke was defined as a deleterious increase in NIH Stroke Scale (NIHSS) score of ≥4 points during hospitalization. We performed multivariate logistic regression analysis and constructed a prediction model based on chart data of 2,398 patients admitted at five medical centers; 203 of the patients had worsening of stroke and 2,186 had not. RESULTS: The results of multivariate logistic regression analysis showed that hemoglobin (odds ratio: 0.529) and albumin (odds ratio: 0.024) were significantly associated with stroke deterioration, as were the modified Rankin Scale on emergency department admission (odds ratio: 4.956) and length of hospitalization (odds ratio: 1.201). After adjusting for age, gender and NIHSS on emergency department admission, only hemoglobin (odds ratio: 0.894, 95% confidence interval: 0.814-0.981, p = 0.018) was associated with worsening. CONCLUSION: Hemoglobin and albumin were found to be risk factors for persistent neurological deterioration during hospitalization following acute ischemic stroke, suggesting that blood viscosity may be related to neurological deterioration.

Diabetic ketoacidosis in children with new-onset type 1 diabetes mellitus: demographics, risk factors and outcome: an 11 year review in Hong Kong.

OBJECTIVES: Diabetic ketoacidosis (DKA) is a life-threatening complication of type 1 diabetes (T1D). The aim of this study is to analyze the incidence, clinical characteristics, management and outcome of children presenting with DKA in new-onset T1D from 2008 to 2018 in Hong Kong. METHODS: Data was extracted from the Hong Kong Childhood Diabetes Registry. All subjects less than 18 years with newly diagnosed T1D from 1 January 2008 to 31 December 2018 managed in the public hospitals were included. Information on demographics, laboratory parameters, DKA-related complications and management were analyzed. RESULTS: In the study period, there were 556 children with newly diagnosed T1D in our registry and 43.3% presented with DKA. The crude incidence rate of new-onset T1D with DKA was 1.79 per 100,000 persons/year (CI: 1.56-2.04). Subjects presenting with DKA were younger (9.5 ± 4.5 vs. 10.5 ± 4.4, p=0.01) and had shorter duration of symptoms (4.2 ± 5.9 days vs. 10.6 ± 17.1 days, p<0.01). Regarding management, up to 12.4% were given insulin boluses and 82.6% were started on insulin infusion 1 h after fluid resuscitation. The rate of cerebral edema was 0.8% and there was no mortality. CONCLUSIONS: Younger age and shorter duration of symptoms were associated with DKA in new-onset T1D. Despite availability of international guidelines, there was inconsistency in acute DKA management. These call for a need to raise public awareness on childhood diabetes as well as standardization of practice in management of pediatric DKA in Hong Kong.

Establishment of electronic chart-based stroke registry system in a medical system in Taiwan.

To establish a prospective, real-time, self-sustainable stroke registry system, we incorporated a registry with an electronic chart to create an electronic chart-based stroke registry system in November 2006. The International Classification of Diseases Ninth Revision code (430-437) was used to auto-enroll stroke patients admitted to neurology departments. Clinical information was written by doctors, nursing information was recorded by nurses, and basic patient information was entered by administrative departments. Numerical data and the date and time of any studies were auto-downloaded from the hospital computer. In total, 212 items were auto-downloaded, including basic patient information, laboratory blood test and examination results, and the date and time of imaging and special intervention. The stroke scales (121 items, National Institutes of Health Stroke Scale, Barthel index, and modified Rankin scale) were designed to be auto-adjusted to reduce incompatibility. The 95 items with pull-down options were used to specify the contents. This registry system can be time-, labor- and money-saving with secured data accuracy.

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs).

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.

The role of ClpX in erythropoietic protoporphyria.

Hemoglobin is an essential biological component of human physiology and its production in red blood cells relies upon proper biosynthesis of heme and globin protein. Disruption in the synthesis of these precursors accounts for a number of human blood disorders found in patients. Mutations in genes encoding heme biosynthesis enzymes are associated with a broad class of metabolic disorders called porphyrias. In particular, one subtype - erythropoietic protoporphyria - is caused by the accumulation of protoporphyrin IX. Erythropoietic protoporphyria patients suffer from photosensitivity and a higher risk of liver failure, which is the principle cause of morbidity and mortality. Approximately 90% of these patients carry loss-of-function mutations in the enzyme ferrochelatase (FECH), while 5% of cases are associated with activating mutations in the C-terminus of ALAS2. Recent work has begun to uncover novel mechanisms of heme regulation that may account for the remaining 5% of cases with previously unknown genetic basis. One erythropoietic protoporphyria family has been identified with inherited mutations in the AAA+ protease ClpXP that regulates ALAS activity. In this review article, recent findings on the role of ClpXP as both an activating unfoldase and degrading protease and its impact on heme synthesis will be discussed. This review will also highlight the role of ClpX dysfunction in erythropoietic protoporphyria.

pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor alpha (HIF-alpha) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.