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Introduction: Pulmonary diseases represent a significant burden to patients and the healthcare system and are one of the leading causes of mortality worldwide. Particularly, the COVID-19 pandemic has had a profound global impact, affecting public health, economies, and daily life. While the peak of the crisis has subsided, the global number of reported COVID-19 cases remains significantly high, according to medical agencies around the world. Furthermore, despite the success of vaccines in reducing the number of deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a gap in the treatment of the disease, especially in addressing uncontrolled inflammation. The massive recruitment of leukocytes to lung tissue and alveoli is a hallmark factor in COVID-19, being essential for effectively responding to the pulmonary insult but also linked to inflammation and lung damage. In this context, mice models are a crucial tool, offering valuable insights into both the pathogenesis of the disease and potential therapeutic approaches. Methods: Here, we investigated the anti-inflammatory effect of the glycosaminoglycan (GAG)-binding chemokine fragment CXCL9(74-103), a molecule that potentially decreases neutrophil transmigration by competing with chemokines for GAG-binding sites, in two models of pneumonia caused by coronavirus infection. Results: In a murine model of betacoronavirus MHV-3 infection, the treatment with CXCL9(74-103) decreased the accumulation of total leukocytes, mainly neutrophils, to the alveolar space and improved several parameters of lung dysfunction 3 days after infection. Additionally, this treatment also reduced the lung damage. In the SARS-CoV-2 model in K18-hACE2-mice, CXCL9(74-103) significantly improved the clinical manifestations of the disease, reducing pulmonary damage and decreasing viral titers in the lungs. Discussion: These findings indicate that CXCL9(74-103) resulted in highly favorable outcomes in controlling pneumonia caused by coronavirus, as it effectively diminishes the clinical consequences of the infections and reduces both local and systemic inflammation.

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The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

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Oreochromis niloticus (tilapia) is one of the most cultivated fish species worldwide. Tilapia farming generates organic waste from fish removal processes in nurseries. Visceral waste can damage natural ecosystems. Therefore, the use of this material as a source of biomolecules helps reduce environmental impacts and improve pharmacological studies. Tilapia viscera were subjected to proteolysis and complexation with an ion-exchange resin. The obtained glycosaminoglycans were purified using ion exchange chromatography (DEAE-Sephacel). The electrophoretic profile and analysis of 1H/13C nuclear magnetic resonance (NMR) spectra allowed for the characterization of the compound as chondroitin sulfate and its sulfation position. This chondroitin was named CST. We tested the ability of CST to reduce leukocyte influx in acute peritonitis models induced by sodium thioglycolate and found a significant reduction in leukocyte migration to the peritoneal cavity, similar to the polymorphonuclear population of the three tested doses of CST. This study shows, for the first time, the potential of CST obtained from O. niloticus waste as an anti-inflammatory drug, thereby contributing to the expansion of the study of molecules with pharmacological functions.

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BACKGROUND: Combined chondroitin sulfate (CS) and glucosamine (GlcN) has been widely used in oral formulations to prevent and treat osteoarthritis. CS is effective for controlling pain in osteoarthritic patients, whereas GlcN can stimulate glycosaminoglycan synthesis, thus reducing extracellular matrix degradation. Although several studies have been published on this topic, the effectiveness of treatment with oral CS and GlcN remains uncertain. The objective of this study was to analyze the progression of experimentally induced osteoarthritis in horses and verify the effectiveness of an oral compound based on CS and GlcN to treat and/or modulate this disease. The study analyzed the metacarpophalangeal joint of the left thoracic limb of 16 horses divided into two groups, with eight horses treated with CS and GlcN in the treated group (GT) and eight untreated horses in the control group (GC). Chondral lesions were induced through arthroscopy, which was defined as time-point zero (T0). Physical, ultrasonographic, and radiographic examinations and synovial fluid biomarkers measurements were performed on days 0, 30, 60, 90, and 120. At the end of the experiment (T4), arthroscopy was performed again to macroscopically evaluate the joints and collect material for microscopic analysis. RESULTS: Significant differences were observed between groups in some evaluated parameters, such as visual lameness assessment, synovial concentrations of prostaglandin E2, and ultrasound examination. However, the GT still presented slightly improved results for joint flexion angle, analysis of lameness using sensors, and histopathological analysis of chondral repair tissue, however, without the statistical significance (p>0.05). CONCLUSIONS: The treatment was considered effective in the clinical modulation of experimental osteoarthritis, with improvement of some parameters in the GT. However, this type of treatment may not be entirely effective to change the catabolic process in articular cartilage and the progressive induced chondral damage.

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Since it is known that hyaluronic acid contributes to soft tissue growth, elasticity, and scar reduction, different strategies of producing HA have been explored in order to satisfy the current demand of HA in pharmaceutical products and formulations. The current interest deals with production via bacterial and yeast fermentation and extraction from animal sources; however, the main challenge is the right extraction technique and strategy since the original sources (e.g., fermentation broth) represent a complex system containing a number of components and solutes, which complicates the achievement of high extraction rates and purity. This review sheds light on the main pathways for the production of HA, advantages, and disadvantages, along with the current efforts in extracting and purifying this high-added-value molecule from different sources. Particular emphasis has been placed on specific case studies attempting production and successful recovery. For such works, full details are given together with their relevant outcomes.

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Prion diseases have been described in humans and other mammals, including sheep, goats, cattle, and deer. Since mice, hamsters, and cats are susceptible to prion infection, they are often used to study the mechanisms of prion infection and conversion. Mammals, such as horses and dogs, however, do not naturally contract the disease and are resistant to infection, while others, like rabbits, have exhibited low susceptibility. Infection involves the conversion of the cellular prion protein (PrPC) to the scrapie form (PrPSc), and several cofactors have already been identified as important adjuvants in this process, such as glycosaminoglycans (GAGs), lipids, and nucleic acids. The molecular mechanisms that determine transmissibility between species remain unclear, as well as the barriers to transmission. In this study, we examine the interaction of recombinant rabbit PrPC (RaPrP) with different biological cofactors such as GAGs (heparin and dermatan sulfate), phosphatidic acid, and DNA oligonucleotides (A1 and D67) to evaluate the importance of these cofactors in modulating the aggregation of rabbit PrP and explain the animal's different degrees of resistance to infection. We used spectroscopic and chromatographic approaches to evaluate the interaction with cofactors and their effect on RaPrP aggregation, which we compared with murine PrP (MuPrP). Our data show that all cofactors induce RaPrP aggregation and exhibit pH dependence. However, RaPrP aggregated to a lesser extent than MuPrP in the presence of any of the cofactors tested. The binding affinity with cofactors does not correlate with these low levels of aggregation, suggesting that the latter are related to the stability of PrP at acidic pH. The absence of the N-terminus affected the interaction with cofactors, influencing the efficiency of aggregation. These findings demonstrate that the interaction with polyanionic cofactors is related to rabbit PrP being less susceptible to aggregation in vitro and that the N-terminal domain is important to the efficiency of conversion, increasing the interaction with cofactors. The decreased effect of cofactors in rabbit PrP likely explains its lower propensity to prion conversion.

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Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.

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PURPOSE: To determine alterations of chondroitin sulfate (CS) that reflect cartilage damage in an experimental osteoarthritis (OA) model as well as in human OA samples. MATERIALS AND METHODS: Rats were subjected to anterior cruciate ligament transection (ACLT; OA) or a sham procedure and sacrificed 14, 28, or 70 days after ACLT for histopathology and analysis of extracted CS. Cartilage samples from 14 patients undergoing hip or shoulder arthroplasty secondary to OA or fracture (control) were subjected to the same protocol. The CS content (µg/mg dry cartilage) after proteolysis was determined by densitometry, using agarose-gel electrophoresis. Molar mass (MM) and peak MM of CS were determined using high-performance size-exclusion chromatography (HPSEC). RESULTS: OA and sham joints at 70 d had 24 [22-24] and 3 [1-6] median histopathology scores, respectively (p < 0.001). Relative CS content (77.7 ± 8.3 µg/mg) was significantly increased in OA samples 70 d after ACLT, as compared to sham (53.5 ± 10.0 µg/mg). Peak MM of CS was higher in OA than in sham samples (P < 0.05). Similarly, CS content and peak MM were higher in cartilage from human OA patients, as compared to fracture samples, reproducing experimental data. CONCLUSION: Cartilage matrix from experimental and human OA samples has increased in the relative CS content. Increase in the peak MM distinguishes CS of the extracellular matrix of OA from normal cartilage.

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BACKGROUND: Stress urinary incontinence carries a significant healthcare burden for women worldwide. Single incision slings are minimally invasive mesh devices designed to treat stress urinary incontinence. For prolapse repair, meshes with higher porosity and lower structural stiffness have been associated with improved outcomes. OBJECTIVE: In this study, we compared the higher stiffness, lower porosity Altis sling with the lower stiffness, higher porosity Solyx sling in an ovine model. We hypothesized that SIS-B would have a negative impact on the host response. STUDY DESIGN: A total of Altis and Solyx single incision slings were implanted suburethrally into sheep according to the manufacturer's instructions on minimal tension. The mesh-urethral-vaginal complex and adjacent ungrafted vagina (no mesh control) were harvested en bloc at 3 months. Masson's trichrome and picrosirius red staining of 6 µm thin sections was performed to measure interfiber distance and tissue integration. Smooth muscle contractility to a 120 mM KCl stimulus was performed in an organ bath to measure myofiber-driven contractions. Standard biochemical assays were used to quantify glycosaminoglycan, total collagen, and elastin content, and collagen subtypes. Bending stiffness was performed in response to a uniaxial force to define susceptibility to folding/buckling. Statistical analysis was performed using Mann-Whitney, Gabriel's pairwise post hoc, Wilcoxon matched-pairs, and chi-square tests. RESULTS: The animals had similar ages (3-5 years), parity (multiparous), and weights (45-72 kg). Trichrome cross sections showed that the Altis sling buckled in a "C" or "S" shape in most samples (8 of 11), whereas buckling after Solyx sling implantation was observed in only a single sample (1 of 13; P=.004). Tissue integration, as measured by the presence of collagen or smooth muscle between the mesh fibers on trichrome 4× imaging, was increased in samples implanted with the Solyx sling compared with the Altis sling (P<.05). Total collagen content decreased significantly with both products when compared with the ungrafted vagina consistent with stress shielding. There was no difference in the 2 groups with regard to glycosaminoglycan or elastin content. The Altis sling mesh tissue complex demonstrated significantly higher amounts of both collagen types I and III than the Solyx sling-implanted tissue and the ungrafted control. Smooth muscle contractility in response to 120 mM KCl was decreased after implantation of both slings compared with the sham (P=.011 and P<.01), with no difference between mesh types (P=.099). Bending stiffness in the Altis sling was more than 4 times lower than in the Solyx, indicating an increased propensity to buckle (0.0186 vs 0.0883). CONCLUSION: The structurally stiffer Altis sling had decreased tissue integration and increased propensity to buckle after implantation. Increased collagen types I and III after the implantation of this device suggests that these changes may be associated with a fibrotic response. In contrast, the Solyx sling largely maintained a flat configuration and had improved tissue integration. The deformation of the Altis sling is not an intended effect and is likely caused by its lower bending stiffness. Both meshes induced a decrease in collagen content and smooth muscle contractility similar to previous findings for prolapse meshes and consistent with stress shielding. The long-term impact of buckling warrants further investigation.

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We report a patient with mucopolysaccharidosis type VI, on long-term enzyme replacement home therapy. Results support the efficacy and safety benefits, with additional advantage of home therapy to minimize the risk of community-transmitted infections.

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Proteoglycans are rapidly emerging as versatile regulators of intracellular catabolic pathways. This is predominantly achieved via the non-canonical induction of autophagy, a fundamentally and evolutionarily conserved eukaryotic pathway necessary for maintaining organismal homeostasis. Autophagy facilitated by either decorin, a small leucine-rich proteoglycan, or perlecan, a basement membrane heparan sulfate proteoglycan, proceeds independently of ambient nutrient conditions. We found that soluble decorin evokes endothelial cell autophagy and breast carcinoma cell mitophagy by directly interacting with vascular endothelial growth factor receptor 2 (VEGFR2) or the Met receptor tyrosine kinase, respectively. Endorepellin, a soluble, proteolytic fragment of perlecan, induces autophagy and endoplasmic reticulum stress within the vasculature, downstream of VEGFR2. These potent matrix-derived cues transduce key biological information via receptor binding to converge upon a newly discovered nexus of core autophagic machinery comprised of Peg3 (paternally expressed gene 3) for autophagy or mitostatin for mitophagy. Here, we give a mechanistic overview of the nutrient-independent, proteoglycan-driven programs utilized for autophagic or mitophagic progression. We propose that catabolic control of cell behavior is an underlying basis for proteoglycan versatility and may provide novel therapeutic targets for the treatment of human disease.

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Abstract Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.

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BACKGROUND Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.

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The close relationship between protein aggregation and neurodegenerative diseases has been the driving force behind the renewed interest in a field where biophysics, neurobiology and nanotechnology converge in the study of the aggregate state. On one hand, knowledge of the molecular principles that govern the processes of protein aggregation has a direct impact on the design of new drugs for high-incidence pathologies that currently can only be treated palliatively. On the other hand, exploiting the benefits of protein aggregation in the design of new nanomaterials could have a strong impact on biotechnology. Here we review the contributions of our research group on novel neuroprotective strategies developed using a purely biophysical approach. First, we examine how doxycycline, a well-known and innocuous antibiotic, can reshape α-synuclein oligomers into non-toxic high-molecular-weight species with decreased ability to destabilize biological membranes, affect cell viability and form additional toxic species. This mechanism can be exploited to diminish the toxicity of α-synuclein oligomers in Parkinson's disease. Second, we discuss a novel function in proteostasis for extracellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in combination with a specific glycosaminoglycan (GAG) present in the extracellular matrix. GAPDH, by changing its quaternary structure from a tetramer to protofibrillar assembly, can kidnap toxic species of α-synuclein, and thereby interfere with the spreading of the disease. Finally, we review a brighter side of protein aggregation, that of exploiting the physicochemical advantages of amyloid aggregates as nanomaterials. For this, we designed a new generation of insoluble biocatalysts based on the binding of photo-immobilized enzymes onto hybrid protein:GAG amyloid nanofibrils. These new nanomaterials can be easily functionalized by attaching different enzymes through dityrosine covalent bonds.

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Mucopolysaccharidosis type I (MPS I) is a lysosomal disorder characterized by a deficiency of alpha-L-iduronidase and storage of undegraded glycosaminoglycans (GAGs). Clinical findings of the disease include heart failure, and patients often need valve replacement. It has been shown that, later in life, MPS I mice develop those abnormalities, but to date, there have not been studies on the progression and pathogenesis of the disease. Therefore, in the present study, we evaluated heart function in normal and MPS I male mice from 2 to 8 months of age. Echocardiographic analysis showed left ventricular enlargement with progressive reduction in ejection fraction, fractional area change, and left ventricular fractional shortening in the MPS I hearts at 6 and 8 months of age and a reduction in acceleration time/ejection time ratio of the pulmonary artery starting at 6 months of age, which suggests pulmonary vascular resistance. Histological and biochemical analysis confirmed progressive GAG storage from 2 months of age and onwards in the myocardium and heart valves, which had also increased in thickness. Additionally, macrophages were present in the MPS I heart tissue. Collagen content was reduced in the MPS I mouse valves. Cathepsin B, an enzyme that is known to be able to degrade collagen and is involved in heart dilatation, displayed a marked elevation in activity in the MPS I mice and could be responsible for the heart dilatation and valves alterations observed. Our results suggest that the MPS I mice have progressive heart failure and valve disease, which may be caused by cathepsin B overexpression.

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The occurrence of restrictive strabismus after conjunctival surgeries is widely described in the literature. Fibrosis causes adherence of the conjunctiva to the Tenon's capsule, intermuscular membrane, adipose orbital tissue, sclera, and extraocular muscle tissue. Fibrosis causes adherence of the conjunctiva to the Tenon's capsule, intermuscular membrane, adipose orbital tissue, sclera, and extraocular muscle tissue. We report a case of the use of a biodegradable collagen-glycosaminoglycan copolymer matrix implant (Ologen®) as an alternative adjuvant, used as a spacer to mechanically separate the subconjunctival and episcleral tissues to prevent fibrosis in a patient undergoing a surgical correction for a strabismus with a restrictive component due to previous surgeries of pterygium excision.

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OBJECTIVES: The objective of this study was to evaluate the use of a chondroitin sulfate and glycosaminoglycan-based chrondro-osseous regenerative compound (CORC) with different local treatments for bone regeneration in dehiscence defects. The hypothesis is that CORC can enhance bone regeneration with or without local treatment. MATERIALS AND METHODS: Twelve mongrel dogs received four implants each in the right femur. Bony defects (4-mm height × 4-mm width) were created and locally treated as follows: reabsorbable membrane (Mem), hidroxyapatite (HA), hydroxyapatite covered with membrane (HA+Mem), or left untreated (Con). Six dogs received one pill of the CORC daily. After 90 days, the implants were retrieved, and histological sections were obtained. The height of bone formation, new bone area (NBA), and bone to implant contact (BIC) within the threads were evaluated to assess the effects of the use of CORC to promote bone regeneration in the defects. Results were statistically analyzed using ANOVA and Tukey's test with 5% significance level. RESULTS: CORC was not capable to increase the height of bone formation, NBA, and BIC. When the local treatments were analyzed regardless of the use of CORC, HA+Mem and Ha presented higher BIC and height of bone formation. There was no difference for NBA among the local treatments. CONCLUSIONS: The hypothesis was rejected since the use of CORC has not increased any of the parameters evaluated. CLINICAL RELEVANCE: Dehiscence-like defects can compromise soft tissue support and result in loss of periodontal health and implants. Hydroxyapatite can induce bone regeneration in the defects created. CORC in the formulation used in this study did not promote further bone regeneration in dehiscence-like defects.

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Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

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Clinical and biochemical improvements are reported on Mucopolysaccharidosis type VI (MPS VI) patients on Enzyme Replacement Therapy (ERT) with rhASB (galsulfase, Naglazyme®), and preclinical and clinical studies have shown clinical benefits of early initiation. We report four unrelated MPS VI children who began ERT as infants (ages 5 days­10 months). The three older patients showed the first clinical signs of MPS VI at baseline, also presenting different degrees of dysostosis multiplex, and two had mild heart disease. The two oldest also had mild facial coarseness, one had hearing conduction deficit and sleep disorder and the other corneal clouding at baseline. After six years on ERT, all four patients have normal urinary GAG values. Although they all showed normal motor and mental development, brain and cervical spine MRI images available from two of the older patients showed abnormalities, while the youngest child continues having normal images. The four patients presented slower progression of bone and joint disease when compared to their affected older siblings. It should be noticed that only two patients in this sample are currently below the 3rd percentile for height: the youngest who has a constitutional factor associated and the eldest who already presented frank dysostosis at 10 months of age. These findings confirm previous studies that report that skeletal features of the disease cannot be completely prevented despite early ERT. Heart disease already present in two of the four infants at baseline got worse over time and appeared in another patient, but the youngest child on ERT introduction still has a normal echocardiogram at six years of age; he also is the only one without corneal clouding after six years follow-up. Our results also suggest that early ERT prevented storage in spleen and liver and may also have improved or prevented progression of facial dysmorphic features, corroborating similar findings seen in previous studies. No safety concerns were identified and none of the patients experienced a serious adverse event. The baseline severity of the disorder of these four infants seems related to age and it is tempting to say that severity on the first year of life is progressive and ERT effectiveness is indirectly related to it. Despite being known that MPS VI progresses differently among patients, the fact that these infants had a slower progression than their older siblings speaks in favor of a very early start of ERT. In conclusion, this report confirms the early manifestations of the disease and provides additional evidence on safety and of the beneficial effects of ERT in patients less than 1 year of age.

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Abstract The purpose of this communication is to indicate a simple and rapid method with a small volume of urine sample to detect urine glycosaminoglycan (GAG) and serve as a screening procedure for mucopolysaccharidoses (MPSs). Total GAG measurement for patients with MPS disorders is considered to be the first step in diagnosis of those heterogeneous group of lysosomal storage disorders presenting clinical phenotype. In this study, modified 9-dimethylmethylene blue method is used for total GAG measurement. Following GAG quantitation, the procedure described here allows GAG isolation from a very a small volume of urine sample and subjected to high-resolution GAG electrophoresis, which can be easily performed in routine clinical diagnostic laboratories. Glycosaminoglycan precipitation is a modified method based on total GAG concentration in the urine. For optimized isolation of total GAG for electrophoresis, instead of considering the urine creatinine concentration, 300 μg/mL GAG containing urine is considered to be the target concentration for the best precipitation with 1000 μL cetylpyridinium chloride (CPC)/citrate buffer. Glycosaminoglycan concentration-based precipitation of urine with CPC allows the laboratory to be able to work with a small volume of urine sample by keeping the precipitating ratio with CPC constant for samples that contain GAG less than 300 μg/mL. Based on the effect of cold buffer using low voltage, GAGs high-resolution electrophoresis banding patterns described here enable a clear separation of keratan sulfate from chondroitin sulfate as well as dermatan sulfate (DS1 and DS2) and heparan sulfate. By this procedure, GAG patterns are more clear, easily identified, and provide a guide for the enzyme analysis deficient in the MPS disorders.