Clinical landscape of citrin deficiency: A global perspective on a multifaceted condition.

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in SLC25A13. The clinical manifestation is very variable and comprises three types: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD: OMIM 605814), post-NICCD including failure to thrive and dyslipidemia caused by citrin deficiency, and adult-onset type II citrullinemia (CTLN2: OMIM 603471). Frequently, NICCD can run with a mild clinical course and manifestations may resolve in the post-NICCD. However, a subset of patients may develop CTLN2 when they become more than 18 years old, and this condition is potentially life-threatening. Since a combination of diet with low-carbohydrate and high-fat content supplemented with medium-chain triglycerides is expected to ameliorate most manifestations and to prevent the progression to CTLN2, early detection and intervention are important and may improve long-term outcome in patients. Moreover, infusion of high sugar solution and/or glycerol may be life-threatening in patients with citrin deficiency, particularly CTLN2. The disease is highly prevalent in East Asian countries but is more and more recognized as a global entity. Since newborn screening for citrin deficiency has only been introduced in a few countries, the diagnosis still mainly relies on clinical suspicion followed by genetic testing or selective metabolic screening. This paper aims at describing (1) the different stages of the disease focusing on clinical aspects; (2) the current published clinical situation in East Asia, Europe, and North America; (3) current efforts in increasing awareness by establishing management guidelines and patient registries, hereby illustrating the ongoing development of a global network for this rare disease.

The therapeutic landscape of citrin deficiency.

Citrin deficiency (CD) is a recessive, liver disease caused by sequence variants in the SLC25A13 gene encoding a mitochondrial aspartate-glutamate transporter. CD manifests as different age-dependent phenotypes and affects crucial hepatic metabolic pathways including malate-aspartate-shuttle, glycolysis, gluconeogenesis, de novo lipogenesis and the tricarboxylic acid and urea cycles. Although the exact pathophysiology of CD remains unclear, impaired use of glucose and fatty acids as energy sources due to NADH shuttle defects and PPARα downregulation, respectively, indicates evident energy deficit in CD hepatocytes. The present review summarizes current trends on available and potential treatments for CD. Baseline recommendation for CD patients is dietary management, often already present as a self-selected food preference, that includes protein and fat-rich food, and avoidance of excess carbohydrates. At present, liver transplantation remains the sole curative option for severe CD cases. Our extensive literature review indicated medium-chain triglycerides (MCT) as the most widely used CD treatment in all age groups. MCT can effectively improve symptoms across disease phenotypes by rapidly supplying energy to the liver, restoring redox balance and inducing lipogenesis. In contrast, sodium pyruvate restored glycolysis and displayed initial preclinical promise, with however limited efficacy in adult CD patients. Ursodeoxycholic acid, nitrogen scavengers and L-arginine treatments effectively address specific pathophysiological aspects such as cholestasis and hyperammonemia and are commonly administered in combination with other drugs. Finally, future possibilities including restoring redox balance, amino acid supplementation, enhancing bioenergetics, improving ureagenesis and mRNA/DNA-based gene therapy are also discussed.

Expression and function of the urea cycle in widely-used hepatic cellular models.

The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.

Distinction of Chaos from Randomness Is Not Possible from the Degree Distribution of the Visibility and Phase Space Reconstruction Graphs.

We investigate whether it is possible to distinguish chaotic time series from random time series using network theory. In this perspective, we selected four methods to generate graphs from time series: the natural, the horizontal, the limited penetrable horizontal visibility graph, and the phase space reconstruction method. These methods claim that the distinction of chaos from randomness is possible by studying the degree distribution of the generated graphs. We evaluated these methods by computing the results for chaotic time series from the 2D Torus Automorphisms, the chaotic Lorenz system, and a random sequence derived from the normal distribution. Although the results confirm previous studies, we found that the distinction of chaos from randomness is not generally possible in the context of the above methodologies.

Pretargeted PET of Osteodestructive Lesions in Dogs.

The last decade has witnessed the creation of a highly effective approach to in vivo pretargeting based on the inverse electron demand Diels-Alder (IEDDA) click ligation between tetrazine (Tz) and trans-cyclooctene (TCO). Despite the steady progression of this technology toward the clinic, concerns have persisted regarding whether this in vivo chemistry will work in humans given their larger size and blood volume. In this work, we describe the use of a 64Cu-labeled Tz radioligand ([64Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted positron emission tomography (PET) imaging of osteodestructive lesions in a large animal model: companion dogs. First, in a small animal pilot study, healthy mice were injected with TCO-BP followed after 1 or 6 h by [64Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [64Cu]Cu-SarAr-Tz, revealing that this approach produced high activity concentrations in the bone (>20 and >15%ID/g in the femur and humerus, respectively, at 24 h post injection) as well as high target-to-background contrast. Subsequently, companion dogs (n = 5) presenting with osteodestructive lesions were administered TCO-BP (5 or 10 mg/kg) followed 1 h later by [64Cu]Cu-SarAr-Tz (2.2-7.3 mCi; 81.4-270.1 MBq). PET scans were collected for each dog 4 h after the administration of the radioligand, and SUV values for the osteodestructive lesions, healthy bones, and kidneys were determined. In these animals, pretargeted PET clearly delineated healthy bone and produced very high activity concentrations in osteodestructive lesions. Low levels of uptake were observed in all healthy organs except for the kidneys and bladder due to the renal excretion of excess radioligand. Ultimately, this work not only illustrates that pretargeted PET with TCO-BP and [64Cu]Cu-SarAr-Tz is an effective tool for the visualization of osteodestructive lesions but also demonstrates for the first time that in vivo pretargeting based on IEDDA click chemistry is feasible in large animals.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Under Pressure: A Comparative Study of Botrytis cinerea Populations from Conventional and Organic Farms in Cyprus and Greece.

The highly heterogeneous nature of Botrytis cinerea provides adaptive benefits to variable environmental regimes. Disentangling pathogen population structure in anthropogenic agroecosystems is crucial to designing more effective management schemes. Herein, we studied how evolutionary forces exerted in different farming systems, in terms of agrochemicals-input, shape B. cinerea populations. In total, 360 B. cinerea isolates were collected from conventional and organic, strawberry and tomato farms in Cyprus and Greece. The occurrence and frequency of sensitivities to seven botryticides were estimated. Results highlighted widespread fungicide resistance in conventional farms since only 15.5% of the isolates were sensitive. A considerable frequency of fungicide-resistant isolates was also detected in the organic farms (14.9%). High resistance frequencies were observed for boscalid (67.7%), pyraclostrobin (67.3%), cyprodinil (65.9%), and thiophanate-methyl (61.4%) in conventional farms, while high levels of multiple fungicide resistance were also evident. Furthermore, B. cinerea isolates were genotyped using a set of seven microsatellite markers (simple sequence repeat [SSR] markers). Index of association analyses (Ia and rBarD) suggest asexual reproduction of the populations, even though the mating-type idiomorphs were equally distributed, indicating frequency-dependent selection. Fungicide resistance was correlated with farming systems across countries and crops, while SSRs were able to detect population structure associated with resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. The expected heterozygosity in organic farms was significantly higher than in conventional, suggesting the absence of selective pressure that may change the allelic abundance in organic farms. However, genetic variance among strawberry and tomato populations was high, ranking host specificity higher than other selection forces studied.

A 3D-Printed Capacitive Smart Insole for Plantar Pressure Monitoring.

Gait analysis refers to the systematic study of human locomotion and finds numerous applications in the fields of clinical monitoring, rehabilitation, sports science and robotics. Wearable sensors for real-time gait monitoring have emerged as an attractive alternative to the traditional clinical-based techniques, owing to their low cost and portability. In addition, 3D printing technology has recently drawn increased interest for the manufacturing of sensors, considering the advantages of diminished fabrication cost and time. In this study, we report the development of a 3D-printed capacitive smart insole for the measurement of plantar pressure. Initially, a novel 3D-printed capacitive pressure sensor was fabricated and its sensing performance was evaluated. The sensor exhibited a sensitivity of 1.19 MPa−1, a wide working pressure range (<872.4 kPa), excellent stability and durability (at least 2.280 cycles), great linearity (R2=0.993), fast response/recovery time (142−160 ms), low hysteresis (DH<10%) and the ability to support a broad spectrum of gait speeds (30−70 steps/min). Subsequently, 16 pressure sensors were integrated into a 3D-printed smart insole that was successfully applied for dynamic plantar pressure mapping and proven able to distinguish the various gait phases. We consider that the smart insole presented here is a simple, easy to manufacture and cost-effective solution with the potential for real-world applications.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

Entropy and Network Centralities as Intelligent Tools for the Investigation of Terrorist Organizations.

In recent years, law enforcement authorities have increasingly used mathematical tools to support criminal investigations, such as those related to terrorism. In this work, two relevant questions are discussed: "How can the different roles of members of a terrorist organization be recognized?" and "are there early signs of impending terrorist acts?" These questions are addressed using the tools of entropy and network theory, more specifically centralities (degree, betweenness, clustering) and their entropies. These tools were applied to data (physical contacts) of four real terrorist networks from different countries. The different roles of the members are clearly recognized from the values of the selected centralities. An early sign of impending terrorist acts is the evolutionary pattern of the values of the entropies of the selected centralities. These results have been confirmed in all four terrorist networks. The conclusion is expected to be useful to law enforcement authorities to identify the roles of the members of terrorist organizations as the members with high centrality and to anticipate when a terrorist attack is imminent, by observing the evolution of the entropies of the centralities.

Competitive Conditions in Global Value Chain Networks: An Assessment Using Entropy and Network Analysis.

We investigated competitive conditions in global value chains (GVCs) for a period of fifteen years (2000-2014), focusing on sector structure, countries' dominance and diversification. For this purpose, we used data from the World Input-Output Database (WIOD) and examined GVCs as weighted directed networks, where countries are the nodes and value added flows are the edges. We compared the in-and out-weighted degree centralization of the sectoral GVC networks in order to detect the most centralized, on the import or export side, respectively (oligopsonies and oligopolies). Moreover, we examined the in- and out-weighted degree centrality and the in- and out-weight entropy in order to determine whether dominant countries are also diversified. The empirical results reveal that diversification (entropy) and dominance (degree) are not correlated. Dominant countries (rich) become more dominant (richer). Diversification is not conditioned by competitiveness.

Vulvar, Perianal and Perineal Cancer After Hidradenitis Suppurativa: A Systematic Review and Pooled Analysis.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory disease that frequently involves the perineal and perianal regions. The association between HS and malignant transformation is a rare but under recognized phenomenon. OBJECTIVE: This systematic review aims to summarize all available cases of vulvar and perianal/perineal cancer emerging in patients with HS, describing clinical and therapeutic particularities of these coexisting conditions in female patients. MATERIALS AND METHODS: This systematic review and pooled analysis was performed in accordance with the PRISMA guidelines; end-of-search date was June 15, 2015. RESULTS: A total of 13 eligible articles were identified; 7 cases of vulvar cancer and 6 cases of perineal/perianal carcinomas in patients with HS were noted. A majority of published cases pertained to rather advanced carcinomas; only occasionally early stage carcinomas were identified. The optimal modifications in the treatment scheme of vulvar, perianal, and perineal cancer in patients with HS have not been established; detailed reporting of recurrence- and survival-related aspects is advised. CONCLUSION: Vulvar, perianal, and perineal cancer represent a rare but serious complication of HS.

Validation of a two-tier grading system in an unselected, consecutive cohort of serous ovarian cancer patients.

PURPOSE: New insights into the carcinogenesis of ovarian cancer (OC) lead to the definition of low-grade and high-grade serous OC. In this study, we validated the MD Anderson Cancer Center (MDACC) two-tier grading system and compared it with the traditional three-tier grading system as suggested by the International Federation of Gynecology and Obstetrics (FIGO). METHODS: Consecutive patients with serous OC were enrolled. These two grading systems were assessed independently from each other. Kaplan-Meier estimates and Cox-regression analyses were performed to validate and compare their prognostic impact. RESULTS: 143 consecutive patients entered the study. According to the Kaplan-Meier estimates, the MDACC grading system (p = 0.001) predicted the progression free survival (PFS) more precisely than the FIGO system (p = 0.025). The MDACC grading system (p = 0.008) but not the FIGO system (p = 0.329) showed a statistically significant difference in terms of disease specific survival (DSS). Multivariable Cox-regression analyses revealed an independent prognostic impact of the MDACC grading system but not of the FIGO system for PFS (HR 1.570; 95 % CI 1.007-2.449; p = 0.047, and HR 0.712; 95 % CI 0.476-1.066; p = 0.099, respectively). Concerning DSS, the two-tier grading system but not the FIGO system showed a prognostic impact in a univariable Cox-regression analysis (HR 2.152; 95 % CI 1.207-3.835; p = 0.009, and HR 1.258; 95 % CI 0.801-1.975; p = 0.319, respectively). CONCLUSIONS: We were able to validate the MDACC grading system in serous OC. Moreover, this grading system was stronger associated with survival than the FIGO system.

Fusarioid keratitis and other superficial infections: A 10-years prospective study from Northeastern Brazil.

BACKGROUND: Fusarium and allied genera (fusarioid) species are common colonizers of roots and aerial plant parts, or act as phytopathogens in forestry and horticultural or grain crops. However, they can also cause a wide range of infections in humans, including onychomycosis, cutaneous and invasive infections. Fusarioid keratitis is characterized by an infection of the cornea with a suppurative and ulcerative appearance, which may cause damage to vision and permanent blindness. The aim of the present study was to investigate the prevalence of fusarioid species, biofilm formation and antifungal susceptibility profiling of clinical isolates recovered from patients with keratitis and dermatomycoses. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed between March, 2012-December, 2022. Demographic, clinical and epidemiological data of patients were also collected. In the present study, most of the patients with keratitis were male (74%), had a median age of 42 years old, worked with plant material or debris and 26% of them reported eye trauma. Regarding dermatomycosis, most of patients were female and exhibited toenail lesions. Forty-seven isolates belonged to the genus Neocosmospora (78.33%), nine to the Fusarium fujikuroi (15%) and four to the Fusarium oxysporum (6.66%) species complexes. Several strains were moderate biofilm producers, specifically among Fusarium annulatum. Most strains showed increased MICs to amphotericin B and ketoconazole and low MICs to itraconazole. MICs ranged from 0.25 to 16 µg/mL for amphotericin B, 0.0625 to >16 µg/mL for ketoconazole and 0.125 to 8 for itraconazole. CONCLUSIONS/SIGNIFICANCE: It is possible to conclude that fusarioid keratitis in Northeastern Brazil is an important and neglected disease, given the high number of cases, increased need for keratoplasty and poor outcome of the disease.

LEMD2-associated progeroid syndrome: Expanding the phenotype of the nuclear envelopathy caused by a defect in LEMD2 gene.

Nuclear envelopathies are rare genetic diseases that compromise the integrity of the nuclear envelope. Patients with a defect in LEM domain nuclear envelope protein 2 (LEMD2) leading to LEMD2-associated progeroid syndrome are exceedingly scarce in number, yet they exhibit shared clinical features including skeletal abnormalities and a prematurely-aged appearance. Our study broadens the understanding of LEMD2-associated progeroid syndrome by detailing its phenotypic and molecular characteristics in the first female and fourth reported case, highlighting a distinct impact on metabolic functions. The patient's history revealed growth delay, facial and skeletal abnormalities, and recurrent abdominal pain crises caused by hepatomegaly. Comparisons with the previously documented cases emphasized similarities in skeletal and facial features while showcasing unique variations, notably in cardiac and hepatic manifestations. In vitro experiments conducted on patient-derived peripheral blood and urinary epithelial cells and LEMD2-downregulated HepG2 cells confirmed abnormalities in the structure of the nuclear envelope in all three tissue-types. Overall, our work offers a comprehensive profile of a patient with LEMD2-related syndrome, emphasizing the hepatic involvement in the disease and broadening our understanding of clinical and molecular implications. This study not only contributes specific insights into LEMD2-related conditions but also underscores potential therapeutic paths for disorders affecting nuclear envelope dynamics.

A Novel Seimatosporium and Other Sporocadaceae Species Associated with Grapevine Trunk Diseases in Cyprus.

Besides well-known grapevine trunk disease (GTD)-related pathogens, there is an increased interest in wood-colonizing fungi that infect grapevines. During 2017-2018, a survey was conducted in Cyprus and wood samples were collected from vines exhibiting typical GTD symptoms. Based on morphological and multilocus phylogenetic analyses (ITS, LSU, bt2, tef1-a), four species in the Sporocadaceae family were described and typified; two in the genus of Seimatosporium: Seim. cyprium sp. nov. and Seim. vitis-viniferae and two in Sporocadus: Spo. kurdistanicus and Spo. rosigena. The teleomorph of Seim. cyprium sp. nov. was also described. Pathogenicity trials with representative isolates of each species were performed on woody stems of two-year-old potted grapevines for 12 months under field conditions. All isolates were pathogenic, causing dark brown to black vascular discoloration, extending upward and downward from the inoculation point. Sporocadus isolates were significantly more aggressive than Seimatosporium with lesion lengths ranging from 9.24 to 6.90 and 4.13 to 4.00 cm, respectively. Successful re-isolations were also evident for all species and isolates. Seim. cyprium sp. nov. is a newly described species, while Spo. kurdistanicus and Spo. rosigena are reported for the first time in Europe on Vitis vinifera, suggesting the potential role of Sporocadaceae in the GTDs complex.

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.