Biomedicines

Mucopolysaccharidosis type II (MPS II; also known as Hunter syndrome) is a rare, inherited lysosomal storage disease. The disease is caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S) due to mutations in the IDS gene, which leads to accumulation of glycosaminoglycans (GAGs). Deficiency of I2S enzyme activity in patients with MPS II leads to progressive lysosomal storage of GAGs in the liver, spleen, heart, bones, joints, and respiratory tract. This process disturbs cellular functioning and leads to multisystemic disease manifestations. Symptoms and their time of onset differ among patients. Diagnosis of MPS II involves assessment of clinical features, biochemical parameters, and molecular characteristics. Life-long enzyme replacement therapy with idursulfase (recombinant human I2S) is the current standard of care. However, an interdisciplinary team of specialists is required to monitor and assess the patient’s condition to ensure optimal care. An increasing number of patients with this rare disease reach adulthood and old age. The transition from pediatric care to the adult healthcare system should be planned and carried out according to guidelines to ensure maximum benefit for the patient. Full article

Recently, research has greatly expanded the knowledge of the endocannabinoid system (ECS) and its involvement in several therapeutic applications. Cannabinoid receptors (CBRs) are present in nearly every mammalian tissue, performing a vital role in different physiological processes (neuronal development, immune modulation, energy homeostasis). The ECS has an essential role in metabolic control and lipid signaling, making it a potential target for managing conditions such as obesity and diabetes. Its malfunction is closely linked to these pathological conditions. Additionally, the immunomodulatory function of the ECS presents a promising avenue for developing new treatments for various types of acute and chronic inflammatory conditions. Preclinical investigations using peripherally restricted CBR antagonists that do not cross the BBB have shown promise for the treatment of obesity and metabolic diseases, highlighting the importance of continuing efforts to discover novel molecules with superior safety profiles. The purpose of this review is to examine the roles of CB1R and CB2Rs, as well as their antagonists, in relation to the above-mentioned disorders. Full article

Despite the recent approval of new therapies, the prognosis for patients with hepatocellular carcinoma (HCC) remains poor. There is a clinical need for new highly effective therapeutic options. Here, we present a combined application of BH3-mimetics as a potential new treatment option for HCC. BH3-mimetics inhibit anti-apoptotic proteins of the BCL-2 family and, thus, trigger the intrinsic apoptosis pathway. Anti-apoptotic BCL-2 proteins such as Bcl-2 and Mcl-1 are frequently overexpressed in HCC. Therefore, we analyzed the efficacy of the two BH3-mimetics ABT-199 (Bcl-2 inhibitor) and MIK665 (Mcl-1 inhibitor) in HCC cell lines with differential expression levels of endogenous Bcl-2 and Mcl-1. While administration of one BH3-mimetic alone did not substantially trigger cell death, the combination of two inhibitors enhanced induction of the intrinsic apoptosis pathway. Both drugs acted synergistically, highlighting the effectivity of this specific BH3-mimetic combination, particularly in HCC cell lines. These results indicate the potential of combining inhibitors of the BCL-2 family as new therapeutic options in HCC. Full article

Background: Infected nonunion of the tibia represents a challenging complication for orthopedic surgeons and poses a major financial burden to healthcare systems. The situation is even more compounded when the nonunion involves the metaphyseal region of long bones, a rare yet demanding complication due to the poor healing potential of infected cancellous bone; this is in addition to the increased likelihood of contamination of adjacent joints. The purpose of this study was to determine the extent and level of evidence in relation to (1) available treatment options for the management of septic tibial metaphyseal nonunions; (2) success rates and bone healing following treatment application; and (3) functional results after intervention. Methods: We searched the MEDLINE, Embase, and CENTRAL databases for prospective and retrospective studies through to 25 January 2021. Human-only studies exploring the efficacy of various treatment options and their results in the setting of septic, quiescent, and metaphyseal (distal or proximal) tibia nonunions in the adult population were included. For infection diagnosis, we accepted definitions provided by the authors of source studies. Of note, clinical heterogeneity rendered data pooling inappropriate. Results: In terms of the species implicated in septic tibial nonunions, staphylococcus aureus was found to be the most commonly isolated microorganism. Many authors implemented the Ilizarov external fixation device with a mean duration of treatment greater than one year. Exceptional or good bone and functional results were recorded in over 80% of patients, although the literature is scarce and possible losses of the follow-up were not recorded. Conclusion: A demanding orthopedic condition that is scarcely studied is infected metaphyseal tibial nonunion. External fixation seems promising, but further research is needed. Systematic Review Registration: PROSPERO No. CRD42020205781. Full article

Kidney diseases can cause severe morbidity, mortality, and health burden. Determining the risk factors associated with kidney damage and deterioration has become a priority for the prevention and treatment of kidney disease. This study followed 497 patients with stage 3–5 chronic kidney disease (CKD) who were treated at the ward of Taipei Veterans General Hospital from January 2006 to 2019 in Taiwan. The patients underwent 3-year-long follow-up sessions for clinical measurements, which occurred every 3 months. Three time-dependent survival models, namely the Cox proportional hazard model (Cox PHM), random survival forest (RSF), and an artificial neural network (ANN), were used to process patient demographics and laboratory data for predicting progression to renal failure, and important features for optimal prediction were evaluated. The individual prediction of CKD progression was validated using the Kaplan–Meier estimation method, based on patients’ true outcomes during and beyond the study period. The results showed that the average concordance indexes for the cross-validation of the Cox PHM, ANN, and RSF models were 0.71, 0.72, and 0.89, respectively. RSF had the best predictive performances for CKD patients within the 3 years of follow-up sessions, with a sensitivity of 0.79 and specificity of 0.88. Creatinine, age, estimated glomerular filtration rate, and urine protein to creatinine ratio were useful factors for predicting the progression of CKD patients in the RSF model. These results may be helpful for instantaneous risk prediction at each follow-up session for CKD patients. Full article

Epigenetic mechanisms are implicated in transcriptional programs driving chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain and extraterminal (BET) proteins, which are epigenetic readers that modulate gene expression. In the phase 3 BETonMACE trial, apabetalone reduced risk of major adverse cardiac events (MACE) by 50% in the CKD subpopulation, indicating favorable effects along the kidney–heart axis. Activation of human renal mesangial cells (HRMCs) to a contractile phenotype that overproduces extracellular matrix (ECM) and inflammatory cytokines, and promotes calcification, frequently accompanies CKD to drive pathology. Here, we show apabetalone downregulated HRMC activation with TGF-β1 stimulation by suppressing TGF-β1-induced α-smooth muscle actin (α-SMA) expression, α-SMA assembly into stress fibers, enhanced contraction, collagen overproduction, and expression of key drivers of fibrosis, inflammation, or calcification including thrombospondin, fibronectin, periostin, SPARC, interleukin 6, and alkaline phosphatase. Lipopolysaccharide-stimulated expression of inflammatory genes IL6, IL1B, and PTGS2 was also suppressed. Transcriptomics confirmed apabetalone affected gene sets of ECM remodeling and integrins. Clinical translation of in vitro results was indicated in CKD patients where a single dose of apabetalone reduced plasma levels of key pro-fibrotic and inflammatory markers, and indicated inhibition of TGF-β1 signaling. While plasma proteins cannot be traced to the kidney alone, anti-fibrotic and anti-inflammatory effects of apabetalone identified in this study are consistent with the observed decrease in cardiovascular risk in CKD patients. Full article

Nuclear factor kappa beta (NF-κB) is a transcriptional factor that plays a crucial role in regulating cancer cell proliferation. Therefore, the inhibition of NF-κB activity by small molecules may be beneficial in cancer therapy. In this report, methyl-thiol-bridged oxadiazole and triazole heterocycles were synthesized via click chemistry and it was observed that the lead structure, 2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c), reduced the viability of MCF-7 cells with an IC₅₀ value of 7.4 µM. Compound 4c also caused concentration-dependent loss of cell viability in chronic myelogenous leukemia (CML) cells. Furthermore, compound 4c inhibited the activation of NF-κB in human CML cells as observed by nuclear translocation and DNA binding assays. Functionally, compound 4c produced PARP cleavage and also suppressed expression of Bcl-2/xl, MMP-9, COX-2, survivin, as well as VEGF, resulting in apoptosis of CML cells. Moreover, ChIP assay showed that compound 4c decreased the binding of COX-2 to the p65 gene promoter. Detailed in silico analysis also indicated that compound 4c targeted NF-κB in CML cells. In conclusion, a novel structure bearing both triazole and oxadiazole moieties has been identified that can target NF-κB in CML cells and may constitute a potential novel drug candidate. Full article

Lipoprotein(a) (Lp(a)) is made up of apoprotein(a) (apo(a)) and an LDL-like particle. The LPA gene encodes apo(a) and thus determines the characteristics and amount of apo(a) and Lp(a). The proportion of Lp(a) in each individual is genetically determined and is only minimally modifiable by the environment or diet. Lp(a) has important pro-atherosclerotic and pro-inflammatory effects. It has been hypothesized that Lp(a) also has pro-coagulant and antifibrinolytic actions. For these reasons, high Lp(a) values are an important independent risk factor for cardiovascular disease and calcific aortic valve stenosis. Numerous studies have been performed in adults about the pathophysiology and epidemiology of Lp(a) and research is under way for the development of drugs capable of reducing Lp(a) plasma values. Much less information is available regarding Lp(a) in children and adolescents. The present article reviews the evidence on this topic. The review addresses the issues of Lp(a) changes during growth, the correlation between Lp(a) values in children and those in their parents, and between Lp(a) levels in children, and the presence of cardiovascular disease in the family. Gaining information on these points is particularly important for deciding whether Lp(a) assay may be useful for defining the cardiovascular risk in children, in order to plan a prevention program early. Full article

Gastrointestinal (GI) complications, including motility disorders, metabolic deficiencies, and changes in gut microbiota following spinal cord injury (SCI), are associated with poor outcomes. After SCI, the autonomic nervous system becomes unbalanced below the level of injury and can lead to severe GI dysfunction. The SmartPill™ is a non-invasive capsule that, when ingested, transmits pH, temperature, and pressure readings that can be used to assess effects in GI function post-injury. Our minipig model allows us to assess these post-injury changes to optimize interventions and ultimately improve GI function. The aim of this study was to compare pre-injury to post-injury transit times, pH, and pressures in sections of GI tract by utilizing the SmartPill™ in three pigs after SCI at 2 and 6 weeks. Tributyrin was administered to two pigs to assess the influences on their gut microenvironment. We observed prolonged GET (Gastric Emptying Time) and CTT (Colon Transit Time), decreases in contraction frequencies (Con freq) in the antrum of the stomach, colon, and decreases in duodenal pressures post-injury. We noted increases in Sum amp generated at 2 weeks post-injury in the colon, with corresponding decreases in Con freq. We found transient changes in pH in the colon and small intestine at 2 weeks post-injury, with minimal effect on stomach pH post-injury. Prolonged GETs and CTTs can influence the absorptive profile in the gut and contribute to pathology development. This is the first pilot study to administer the SmartPill™ in minipigs in the context of SCI. Further investigations will elucidate these trends and characterize post-SCI GI function. Full article

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC), first to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxycytosine (5caC). Evidence suggests that changes in TET expression may impact cell function and the phenotype of aging. Proliferation, apoptosis, markers of autophagy and double-strand DNA break repair, and the expression of Fibulin 5 were assessed by flow cytometry in TET1 and TET2-overexpressing fibroblasts isolated from sun-unexposed skin of young (23–35 years) and age-advanced (75–94 years) individuals. In cells derived from young individuals, TET1 overexpression resulted in the inhibition of proliferation and apoptosis by 37% (p = 0.03) and 24% (p = 0.05), respectively, while the overexpression of TET2 caused a decrease in proliferation by 46% (p = 0.01). Notably, in cells obtained from age-advanced individuals, TETs exhibited different effects. Specifically, TET1 inhibited proliferation and expression of autophagy marker Beclin 1 by 45% (p = 0.05) and 28% (p = 0.048), respectively, while increasing the level of γH2AX, a marker of double-strand DNA breaks necessary for initiating the repair process, by 19% (p = 0.04). TET2 inhibited proliferation by 64% (p = 0.053) and increased the level of γH2AX and Fibulin 5 by 46% (p = 0.007) and 29% (p = 0.04), respectively. These patterns of TET1 and TET2 effects suggest their involvement in regulating various fibroblast functions and that some of their biological actions depend on the donor’s age. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the etiologic agent for the pneumonia outbreak that started in early December 2019 in Wuhan City, Hubei Province, China. To date, coronavirus disease (COVID-19) has caused almost 6 million deaths worldwide. The ability to propagate the virus into a customizable volume will enable better research on COVID-19 therapy, vaccine development, and many others. In the search for the most efficient replication host, we inoculated three (3) local SARS-CoV-2 isolates of different lineages (Clade L/Lineage B Wuhan, Clade GR/Lineage B.1.1.354, and Clade O/Lineage B.6.2) into various clinically important mammalian cell lines. The replication profile of these isolates was evaluated based on the formation of cytopathic effects (CPE), viral load (Ct value and plaque-forming unit (pfu)), as well as observation by electron microscopy (EM). Next-generation sequencing (NGS) was performed to examine the genomic stability of the propagated SARS-CoV-2 in these cell lines. Our study found that Vero E6 and Vero CCL-81 cell lines posed similar capacities in propagating the local isolates, with Vero CCL-81 demonstrating exceptional potency in conserving the genomic stability of the Lineage B Wuhan isolate. In addition, our study demonstrated the utility of Calu-3 cells as a replication host for SARS-CoV-2 without causing substantial cellular senescence. In conclusion, this study provides crucial information on the growth profile of Malaysian SARS-CoV-2 in various mammalian cell lines and thus will be a great source of reference for better isolation and propagation of the SARS-CoV-2 virus isolated in Malaysia. Full article

Gentian violet (GV) is known to have antibacterial and antifungal effects, but recent studies have demonstrated its inhibitory effects on the growth of several types of cancer cells. Here, we investigated the anticancer efficacy of GV in ovarian cancer cells. GV significantly reduced the proliferation of OVCAR8, SKOV3, and A2780 cells. Results of transferase dUTP nick and labeling (TUNEL) assay and Western blot assay indicated that the inhibitory effect of GV on ovarian cancer cells was due to the induction of apoptosis. Moreover, GV significantly increased reactive oxygen species (ROS) and upregulated the expression of p53, PUMA, BAX, and p21, critical components for apoptosis induction, in ovarian cancer cells. Our results suggest that GV is a novel antiproliferative agent and is worthy of exploration as a potential therapeutic agent for ovarian cancer. Full article

Gastroesophageal reflux disease (GERD) has been growing globally, with an increasing burden on the healthcare system due to multiple factors, such as aging and obesity. The current study evaluated the feasibility of endoscopic balloon-assisted laser treatment (EBLT) in a porcine model. GERD was initially developed in three animals via botulinum toxin injection into lower esophageal sphincter (LES). A week after the injection, the EBLT was performed on the GERD-developed models (control = 1 vs. treated = 2). A dose of 30 W of 980 nm laser light was endoscopically applied for 90 s to the LES. Both endoscopic ultrasound and manometry were performed before and after the EBLT. After 12 weeks, esophageal tissues were extracted and prepared for histological analysis. The maximum mucosa temperature was below 50 °C during the EBLT. Compared to control, the treated group yielded thicker and shorter LES muscle layers and maintained LES pressure. Through histology, the EBLT reinforced the muscularis layer with preserved mucosa and mild remodeling of the intermuscular collagen in the LES. The current study demonstrated the feasibility of EBLT as a new endoscopic approach for GERD. Further studies will examine the EBLT in a larger number of animals to warrant efficacy and safety for clinical translations Full article

Regulation of the human IGF2 gene displays multiple layers of control, which secures a genetically and epigenetically predetermined gene expression pattern throughout embryonal growth and postnatal life. These predominantly nuclear regulatory mechanisms converge on the function of the IGF2-H19 gene cluster on Chromosome 11 and ultimately affect IGF2 gene expression. Deregulation of such control checkpoints leads to the enhancement of IGF2 gene transcription and/or transcript stabilization, ultimately leading to IGF-II peptide overproduction. This type of anomaly is responsible for the effects observed in terms of both abnormal fetal growth and increased cell proliferation, typically observed in pediatric overgrowth syndromes and cancer. We performed a review of relevant experimental work on the mechanisms affecting the human IGF2 gene at the epigenetic, transcriptional and transcript regulatory levels. The result of our work, indeed, provides a wider and diversified scenario for IGF2 gene activation than previously envisioned by shedding new light on its extended regulation. Overall, we focused on the functional integration between the epigenetic and genetic machinery driving its overexpression in overgrowth syndromes and malignancy, independently of the underlying presence of loss of imprinting (LOI). The molecular landscape provided at last strengthens the role of IGF2 in cancer initiation, progression and malignant phenotype maintenance. Finally, this review suggests potential actionable targets for IGF2 gene- and regulatory protein target-degradation therapies. Full article

Chronic kidney disease (CKD) is a significant global public health concern associated with high morbidity and mortality rates. The maintenance of oxalate homeostasis plays a critical role in preserving kidney health, particularly in the context of CKD. Although the relationship between oxalate and kidney stone formation has been extensively investigated, our understanding of oxalate homeostasis in non-stone-forming CKD remains limited. This review aims to present an updated analysis of the existing literature, focusing on the intricate mechanisms involved in oxalate homeostasis in patients with CKD. Furthermore, it explores the key factors that influence oxalate accumulation and discusses the potential role of oxalate in CKD progression and prognosis. The review also emphasizes the significance of the gut–kidney axis in CKD oxalate homeostasis and provides an overview of current therapeutic strategies, as well as potential future approaches. By consolidating important findings and perspectives, this review offers a comprehensive understanding of the present knowledge in this field and identifies promising avenues for further research. Full article

Obesity and insulin resistance are associated with the inflamed and defective adipose tissue (AT) phenotype, and are established risk factors for cardiovascular diseases (CVDs). Extracellular vesicles (EVs) are a heterogeneous group of cell-derived lipid membrane vesicles involved in the onset and development of many pathologies, including insulin resistance, diabetes, and CVDs. The inflammation associated with overweight and obesity triggers the transition of the AT secretome from healthy to pathological, with a consequent increased expression of pro-inflammatory mediators. Epicardial adipose tissue (EAT) is a specialized fat depot that surrounds the heart, in direct contact with the myocardium. Recently, the role of EAT in regulating the physiopathology of many heart diseases has been increasingly explored. In particular, the EAT phenotype and derived EVs have been associated with the onset and exacerbation of CVDs. In this review, we will focus on the role of the AT secretome in the case of CVDs, and will discuss the beneficial effects of EVs released by AT as promising therapeutic candidates. Full article

Propentofylline (PROP) is a methylated xanthine compound that diminishes the activation of microglial cells and astrocytes, which are neuronal cells strongly associated with many neurodegenerative diseases. Based on previously observed remyelination and neuroprotective effects, PROP has also been proposed to increment antioxidant defenses and to prevent oxidative damage in neural tissues. Since most neurodegenerative processes have free radicals as molecular pathological agents, the aim of this study was to evaluate the antioxidant effects of 12.5 mg·kg⁻¹·day⁻¹ PROP in plasma and the brainstem of Wistar rats exposed to the gliotoxic agent 0.1% ethidium bromide (EB) for 7–31 days. The bulk of the data here demonstrates that, after 7 days of EB treatment, TBARS levels were 2-fold higher in the rat CNS than in control, reaching a maximum of 2.4-fold within 15 days. After 31 days of EB treatment, lipoperoxidation in CNS was still 65% higher than that in the control. Clearly, PROP treatment limited the progression of lipoperoxidation in EB-oxidized CNS: it was, for example, 76% lower than in the EB-treated group after 15 days. Most of these effects were associated with PROP-induced activity of glutathione reductase in the brainstem: the EB + PROP group showed 59% higher GR activity than that of the EB or control groups within 7 days. In summary, aligning with previous studies from our group and with literature about MTXs, we observed that propentofylline (PROP) improved the thiol-based antioxidant defenses in the rat brainstem by the induction of the enzymatic activity of glutathione reductase (GR), which diminished lipid oxidation progression and rebalanced the redox status in the CNS. Full article

The presence of COVID-19 antibodies in the maternal circulation is assumed to be protective for newborns against SARS-CoV-2 infection. We investigated whether maternal COVID-19 antibodies crossed the transplacental barrier and whether there was any difference in the hematological parameters of neonates born to mothers who recovered from COVID-19 during pregnancy. The cross-sectional study was conducted at the Saidu Group of Teaching Hospitals, located in Swat, Khyber Pakhtunkhwa. After obtaining written informed consent, 115 healthy, unvaccinated mother-neonate dyads were included. A clinical history of COVID-19-like illness, laboratory-confirmed diagnosis, and contact history were obtained. Serum samples from mothers and neonates were tested for SARS-CoV-2 anti-receptor-binding domain (anti-RBD) IgG antibodies. Hematological parameters were assessed with complete blood counts (CBC) and peripheral blood smear examinations. The study population consisted of 115 mothers, with a mean age of 29.44 ± 5.75 years, and most women (68/115 (59.1%)) were between 26 and 35 years of age. Of these mothers, 88/115 (76.5 percent) tested positive for SARS-CoV-2 anti-RBD IgG antibodies, as did 83/115 (72.2 percent) neonatal cord blood samples. The mean levels of SARS-CoV-2 IgG antibodies in maternal and neonatal blood were 19.86 ± 13.82 (IU/mL) and 16.16 ± 12.90 (IU/mL), respectively, indicating that maternal antibodies efficiently crossed the transplacental barrier with an antibody transfer ratio of 0.83. The study found no significant difference in complete blood count (CBC) parameters between seropositive and seronegative mothers, nor between neonates born to seropositive and seronegative mothers. Full article

Transplantation is a clinical procedure that treats a variety of diseases yet is unattainable for many patients due to a nationwide organ shortage and the harsh side effects of chronic immune suppression. Xenografted pig organs are an attractive alternative to traditional allografts and would provide an endless supply of transplantable tissue, but transplants risk rejection by the recipient’s immune system. An essential component of the rejection immune response is the complement system. Sertoli cells, an immunoregulatory testicular cell, survive complement as xenografts long term without any immune suppressants. We hypothesized that exposure to the xenogeneic complement influences Sertoli cell gene expression of other accommodation factors that contribute to their survival; thus, the purpose of this study was to describe these potential changes in gene expression. RNA sequencing of baseline neonatal pig Sertoli cells (NPSC) as compared to NPSC after exposure to normal human serum (NHS, containing complement) revealed 62 significantly differentially expressed genes (DEG) that affect over 30 pathways involved in immune regulation, cell survival, and transplant accommodation. Twelve genes of interest were selected for further study, and Sertoli cell protein expression of CCL2 and the accommodation factor A20 were confirmed for the first time. Functional pathway analyses were conducted in NPSC and three biological clusters were revealed as being considerably affected by NHS exposure: innate immune signaling, cytokine signaling, and T cell regulation. Better understanding of the interaction of Sertoli cells with complement in a xenograft environment may reveal the mechanisms behind immune-privileged systems to increase graft viability. Full article