Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Dyssegmental dysplasia Rolland-Desbuquois type is caused by pathogenic variants in HSPG2 - a founder haplotype shared in five patients.

Dyssegmental dysplasia (DD) is a severe skeletal dysplasia comprised of two subtypes: lethal Silverman-Handmaker type (DDSH) and nonlethal Rolland-Desbuquois type (DDRD). DDSH is caused by biallelic pathogenic variants in HSPG2 encoding perlecan, whereas the genetic cause of DDRD remains undetermined. Schwartz-Jampel syndrome (SJS) is also caused by biallelic pathogenic variants in HSPG2 and is an allelic disorder of DDSH. In SJS and DDSH, 44 and 8 pathogenic variants have been reported in HSPG2, respectively. Here, we report that five patients with DDRD carried four pathogenic variants in HSPG2: c.9970 G > A (p.G3324R), c.559 C > T (p.R187X), c7006 + 1 G > A, and c.11562 + 2 T > G. Two patients were homozygous for p.G3324R, and three patients were heterozygous for p.G3324R. Haplotype analysis revealed a founder haplotype spanning 85,973 bp shared in the five patients. SJS, DDRD, and DDSH are allelic disorders with pathogenic variants in HSPG2.

Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

SARS-CoV-2 variants evolve to rely more on heparan sulfate (HS) for viral attachment and subsequent infection. In our earlier work, we demonstrated that the Delta variant's spike protein binds more strongly to HS compared to WT SARS-CoV-2, leading to enhanced cell internalization via syndecans (SDCs), a family of transmembrane HS proteoglycans (HSPGs) facilitating the cellular entry of the original strain. Using our previously established ACE2- or SDC-overexpressing cellular models, we now compare the ACE2- and SDC-dependent cellular uptake of heat-inactivated WT SARS-CoV-2 with the Delta and Omicron variants. Internalization studies with inactivated virus particles showed that ACE2 overexpression could not compensate for the loss of HS in Omicron's internalization, suggesting that this variant primarily uses HSPGs to enter cells. Although SDCs increased the internalization of all three viruses, subtle differences could be detected between their SDC isoform preferences. The Delta variant particularly benefitted from SDC1, 2, and 4 overexpression for cellular entry, while SDC4 had the most prominent effect on Omicron internalization. The SDC4 knockdown (KD) in Calu-3 cells reduced the cellular uptake of all three viruses, but the inhibition was the most pronounced for Omicron. The polyanionic heparin also hindered the cellular internalization of all three viruses with a dominant inhibitory effect on Omicron. Omicron's predominant HSPG affinity, combined with its preference for the universally expressed SDC4, might account for its efficient transmission yet reduced pathogenicity.

Impact of the heparan sulfate proteoglycan perlecan on human disease and health.

Perlecan, a basement membrane-type heparan sulfate proteoglycan, is an important molecule in the functional diversity of organisms because of the diversity of its glycan chains and the multifunctionality of its core proteins. Human diseases associated with perlecan have been identified using gene-deficient mice. Two human diseases related to perlecan have been reported. One is Silverman-Handmaker type dyssegmental dysplasia, resulting from the complete loss of function of the HSPG2 gene that encodes perlecan core protein, which is mapped to chromosome 1p36. The other is Schwartz-Jampel syndrome resulting from the partial loss of function of the HSPG2 gene. Subsequent in vivo and in vitro studies have revealed the organ-specific functions of perlecan, suggesting its involvement in the pathogenesis of various human diseases. In this review, we discuss the role of perlecan in human diseases and summarize our knowledge about perlecan as a future therapeutic target to treat related diseases and for healthy longevity.

DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2).

DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-ß-cyclodextrin (MßCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells.

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

Heparan sulfate proteoglycan expression in the regenerating zebrafish fin.

BACKGROUND: Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)-anchored proteins, and transmembrane-anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown. RESULTS: Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions. CONCLUSIONS: HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration.

Two different sources of Perlecan cooperate for its function in the basement membrane of the Drosophila wing imaginal disc.

BACKGROUND: The basement membrane (BM) provides mechanical shaping of tissues during morphogenesis. The Drosophila BM proteoglycan Perlecan is vital for this process in the wing imaginal disc. This function is thought to be fostered by the heparan sulfate chains attached to the domain I of vertebrate Perlecan. However, this domain is not present in Drosophila, and the source of Perlecan for the wing imaginal disc BM remains unclear. Here, we tackle these two issues. RESULTS: In silico analysis shows that Drosophila Perlecan holds a domain I. Moreover, by combining in situ hybridization of Perlecan mRNA and protein staining, together with tissue-specific Perlecan depletion, we find that there is an autonomous and a non-autonomous source for Perlecan deposition in the wing imaginal disc BM. We further show that both sources cooperate for correct distribution of Perlecan in the wing imaginal disc and morphogenesis of this tissue. CONCLUSIONS: These results show that Perlecan is fully conserved in Drosophila, providing a valuable in vivo model system to study its role in BM function. The existence of two different sources for Perlecan incorporation in the wing imaginal disc BM raises the possibility that inter-organ communication mediated at the level of the BM is involved in organogenesis.

Extracellular matrix scaffolding in angiogenesis and capillary homeostasis.

The extracellular matrix (ECM) of blood vessels, which is composed of both the vascular basement membrane (BM) and the interstitial ECM is identified as a crucial component of the vasculature. We here focus on the unique molecular composition and scaffolding of the capillary ECM, which provides structural support to blood vessels and regulates properties of endothelial cells and pericytes. The major components of the BM are collagen IV, laminins, heparan sulfate proteoglycans and nidogen and also associated proteins such as collagen XVIII and fibronectin. Their organization and scaffolding in the BM is required for proper capillary morphogenesis and maintenance of vascular homeostasis. The BM also regulates vascular mechanosensing. A better understanding of the mechanical and structural properties of the vascular BM and interstitial ECM therefore opens new perspectives to control physiological and pathological angiogenesis and vascular homeostasis. The overall aim of this review is to explain how ECM scaffolding influences angiogenesis and capillary integrity.

Endorepellin evokes an angiostatic stress signaling cascade in endothelial cells.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway consisting of PERK, eIF2α, ATF4, and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2, which, in turn, phosphorylated PERK at Thr980 Subsequently, PERK phosphorylated eIF2α at Ser51, upregulating its downstream effector proteins ATF4 and GADD45α. RNAi-mediated knockdown of PERK or eIF2α abrogated the endorepellin-mediated up-regulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2-4 h activated PERK and induced GADD45α vis à vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin. Notably, chronic exposure of aortic rings to endorepellin for 7-9 days markedly suppressed vessel sprouting, an angiostatic effect that was rescued by blocking PERK kinase activity. Our findings unravel a mechanism by which an extracellular matrix protein evokes stress signaling in endothelial cells, which leads to angiostasis.

Heparan sulfate proteoglycan molecules, syndecan and perlecan, have distinct roles in the maintenance of Drosophila germline stem cells.

The Drosophila female germline stem cell (GSC) niche provides an excellent model for understanding the stem cell niche in vivo. The GSC niche is composed of stromal cells that provide growth factors for the maintenance of GSCs and the associated extracellular matrix (ECM). Although the function of stromal cells/growth factors has been well studied, the function of the ECM in the GSC niche is largely unknown. In this study, we investigated the function of syndecan and perlecan, molecules of the heparan sulfate proteoglycan (HSPG) family, as the main constituents of the ECM. We found that both of these genes were expressed in niche stromal cells, and knockdown of them in stromal cells decreased GSC number, indicating that these genes are important niche components. Interestingly, our genetic analysis revealed that the effects of syndecan and perlecan on the maintenance of GSC were distinct. While the knockdown of perlecan in the GSC niche increased the number of cystoblasts, a phenotype suggestive of delayed differentiation of GSCs, the same was not true in the context of syndecan. Notably, the overexpression of syndecan and perlecan did not cause an expansion of the GSC niche, opposing the results reported in the context of glypican, another HSPG gene. Altogether, our data suggest that HSPG genes contribute to the maintenance of GSCs through multiple mechanisms, such as the control of signal transduction, and ligand distribution/stabilization. Therefore, our study paves the way for a deeper understanding of the ECM functions in the stem cell niche.

Gene co-expression and alternative splicing analysis of key metabolic tissues to unravel the regulatory signatures of fatty acid composition in cattle.

Increasing the healthy/unhealthy fatty acid (FA) ratio in meat is one of the urgent tasks required to address consumer concerns. However, the regulatory mechanisms ultimately resulting in FA profiles vary among animals and remain largely unknown. In this study, using ~1.2 Tb high-quality RNA-Seq-based transcriptomic data of 188 samples from four key metabolic tissues (rumen, liver, muscle, and backfat) together with the contents of 49 FAs in backfat, the molecular regulatory mechanisms of these tissues contributing to FA formation in cattle were explored. Using this large dataset, the alternative splicing (AS) events, one of the transcriptional regulatory mechanisms in four tissues were identified. The highly conserved and absent AS events were detected in rumen tissue, which may contribute to its functional differences compared with the other three tissues. In addition, the healthy/unhealthy FA ratio related AS events, differential expressed (DE) genes, co-expressed genes, and their functions in four tissues were analysed. Eight key genes were identified from the integrated analysis of DE, co-expressed, and AS genes between animals with high and low healthy/unhealthy FA ratios. This study provides an applicable pipeline for AS events based on comprehensive RNA-Seq analysis and improves our understanding of the regulatory mechanism of FAs in beef cattle.

Expression and Circulating Levels of Perlecan in Breast Cancer - Implications for Oestrogen Dependent Stromal Remodeling.

Localised breast cancer can be cured by surgery and adjuvant treatments, but mortality remains high as some tumours metastasize early. Perlecan is a basement membrane (BM) protein involved in tumour development and progression. Here, mRNA and protein expression of perlecan, and mRNA expression of matrix degrading enzymes were studied in normal breast and invasive breast cancer, and correlated to prognostic risk factors, in particular oestrogen status. Moreover, plasma levels of perlecan were measured in patients with breast cancer and compared with controls. mRNA data was extracted from the Cancer Genome Atlas database. Perlecan protein expression was visualized using immunofluorescence and plasma levels measured by ELISA assay. Perlecan mRNA levels were twice as high in normal breast compared with breast cancer tissue. A strong correlation was found between mRNA expression of perlecan and several matrix-degrading enzymes in oestrogen receptor positive (ER+) tumours. Perlecan protein was localized to both epithelial and vascular BMs, but absent in the stroma in normal breast. In breast cancer, the expression of perlecan in epithelial BM was fragmented or completely lost, with a marked upregulation of perlecan expression in the stroma. Significantly higher levels of perlecan were found in plasma of ER+ patients when compared with ER- patients. This study shows that perlecan expression and degradation in breast cancer may be linked to the ER status of the tumour.

Elevated hypertrophy, growth plate maturation, glycosaminoglycan deposition, and exostosis formation in the Hspg2 exon 3 null mouse intervertebral disc.

Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of receptors directing cellular proliferation, differentiation, and assembly of extracellular matrix. Perlecan HS/CS-mediated interactions promote extracellular matrix assembly/stabilisation and tissue functional properties, and thus, removal of perlecan HS/CS should affect extracellular matrix function and homeostasis. Hspg2 exon 3 null intervertebral discs accumulated significantly greater glycosaminoglycan in the nucleus pulposus, annulus fibrosus, and vertebral growth plates than C57BL/6 wild-type (WT) I intervertebral discs. Proliferation of intervertebral disc progenitor cells was significantly higher in Hspg2 exon 3 null intervertebral discs, and these cells became hypertrophic by 12 weeks of age and were prominent in the vertebral growth plates but had a disorganised organisation. C57BL/6 WT vertebral growth plates contained regular columnar growth plate chondrocytes. Exostosis-like, ectopic bone formation occurred in Hspg2 exon 3 null intervertebral discs, and differences were evident in disc cell maturation and in matrix deposition in this genotype, indicating that perlecan HS/CS chains had cell and matrix interactive properties which repressively maintained tissue homeostasis in the adult intervertebral disc.

ADAMTS1 and HSPG2 mRNA levels in cumulus cells are related to human oocyte quality and controlled ovarian hyperstimulation outcomes.

PURPOSE: The study investigated potential correlations between the expression levels of ADAMTS1 and HSPG2 in cumulus cells (CCs) and controlled ovarian hyperstimulation (COH) outcomes. METHODS: RT-PCR was used to determine ADAMTS1 and HSPG2 mRNA levels in mice CCs at different timepoints (0, 4, 8, 12, and 16 h) after human chorionic gonadotropin (hCG) injection, and in CCs after RNAi treatment. Women with polycystic ovary syndrome (PCOS) (n = 45) and normal ovulatory controls (n = 103) undergoing IVF/ICSI were recruited. Relative ADAMTS1 and HSPG2 mRNA levels were measured by RT-PCR. Moreover, correlations of ADAMTS1 and HSPG2 levels with COH outcomes were analyzed. RESULTS: At different timepoints after hCG treatment, ADAMTS1 mRNA had the highest level at 12 h, whereas HSPG2 showed opposite profiles to ADAMTS1 with the lowest level at 12 h. HSPG2 expression was upregulated after ADAMTS1 RNAi treatment The PCOS group had higher HSPG2 and lower ADAMTS1 expression levels than controls. In normal ovulatory women (control group), a higher expression of ADAMTS1 and lower expression of HSPG2 were associated with more mature oocytes, transplantable embryos, and good quality embryos, whereas higher transplantable embryo rates and good quality embryo rates were obtained only with lower HSPG2 expression. ROC curves showed the co-measurement of ADAMTS1 and HSPG2 had a better predictive power than separate analyses. CONCLUSION: The dynamic profiles of ADAMTS1 and HSPG2 were inversely correlated in CCs. In PCOS and normal ovulatory patients, higher ADAMTS1 and lower HSPG2 expression levels in CCs were related to better COH outcomes.

The extracellular matrix proteoglycan perlecan facilitates transmembrane semaphorin-mediated repulsive guidance.

The Drosophila transmembrane semaphorin-1a (Sema-1a) is a repulsive guidance cue that uses the Plexin A (PlexA) receptor during neural development. Sema-1a is required in axons to facilitate motor axon defasciculation at guidance choice points. We found that mutations in the trol gene strongly suppress Sema-1a-mediated repulsive axon guidance. trol encodes the phylogenetically conserved secreted heparan sulfate proteoglycan (HSPG) perlecan, a component of the extracellular matrix. Motor axon guidance defects in perlecan mutants resemble those observed in Sema-1a- and PlexA-null mutant embryos, and perlecan mutants genetically interact with PlexA and Sema-1a. Perlecan protein is found in both the CNS and the periphery, with higher expression levels in close proximity to motor axon trajectories and pathway choice points. Restoring perlecan to mutant motor neurons rescues perlecan axon guidance defects. Perlecan augments the reduction in phospho-focal adhesion kinase (phospho-FAK) levels that result from treating insect cells in vitro with Sema-1a, and genetic interactions among integrin, Sema-1a, and FAK in vivo support an antagonistic relationship between Sema-1a and integrin signaling. Therefore, perlecan is required for Sema-1a-PlexA-mediated repulsive guidance, revealing roles for extracellular matrix proteoglycans in modulating transmembrane guidance cue signaling during neural development.

Inhibition of Human Cytomegalovirus Entry into Host Cells Through a Pleiotropic Small Molecule.

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.

Early infantile-onset epileptic encephalopathy 28 due to a homozygous microdeletion involving the WWOX gene in a region of uniparental disomy.

The genetic etiologies of many rare disorders, including early infantile epileptic encephalopathies, are largely undiagnosed. A 6-year-old girl was admitted to the National Institutes of Health Undiagnosed Diseases Program with profound intellectual disability, infantile-onset seizures, chronic respiratory failure, facial dysmorphisms, skeletal abnormalities, and atrial septum defect. A large region of homozygosity was discovered on chromosome 16, spanning 16q22.1-16q24.3' caused by uniparental disomy (UPD) that included a maternally inherited homozygous microdeletion covering exon 6 of WWOX (NM_016373.3). mRNA expression analysis revealed that the deletion led to nonsense-mediated decay of the NM_016373.3 transcript; the exon 6 of an alternative transcript (NM_130791.3), lacking the short-chain dehydrogenase, was utilized. The microdeletion in WWOX explains the seizures and intellectual disability, while pathogenic variants in another gene, HSPG2, are likely responsible for the patient's skeletal abnormalities. This report describes a rare autosomal recessive disorder with multiple genetic etiologies, one of which involves UPD.