Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies.

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.

Neuropsychological Profile of Hospitalized Patients Due to COVID-19: Clinical and Inflammatory Correlates.

OBJECTIVES: In the present study, we investigated the pattern of cognitive difficulties in hospitalized patients due to COVID-19 and its relation with the clinical features of the disease. METHOD: Forty hospitalized patients with COVID-19 [mean age: 46.98 years (SD = 9.30); mean years of education: 13.65 (SD = 2.07) and 40 sex-, age- and education-matched healthy controls completed a set of neuropsychological measures administered by telephone. Participants' premorbid intellectual skills and patients' anxiety and depressive symptoms were also evaluated. The association of COVID-19-related biomarkers [oxygen saturation (SpO2), C-reactive protein (CRP), D-dimer and ferritin levels] with neuropsychological performances was examined with a series of hierarchical multiple linear regression analyses, after controlling for demographic and clinical characteristics, psychological distress and premorbid intellectual skills. RESULTS: Patients performed worse than healthy participants on measures of verbal memory, attention and working memory. SpO2 levels were associated with patients' performance on verbal and working memory, whereas CRP levels were associated with performance on verbal memory, abstract reasoning and verbal fluency, after controlling for demographic and clinical characteristics. Ferritin levels predicted performance on the verbal fluency test, whereas D-dimer levels did not predict any of the neuropsychological measures. CONCLUSIONS: Cognitive difficulties in verbal memory, attention and working memory were noted in patients with COVID-19. Markers of hyperinflammation predicted patients' performance above and beyond demographic characteristics, duration of symptoms, length of hospitalization and psychological distress.

Saccharina latissima, candy-factory waste, and digestate from full-scale biogas plant as alternative carbohydrate and nutrient sources for lactic acid production.

To substitute petroleum-based materials with bio-based alternatives, microbial fermentation combined with inexpensive biomass is suggested. In this study Saccharina latissima hydrolysate, candy-factory waste, and digestate from full-scale biogas plant were explored as substrates for lactic acid production. The lactic acid bacteria Enterococcus faecium, Lactobacillus plantarum, and Pediococcus pentosaceus were tested as starter cultures. Sugars released from seaweed hydrolysate and candy-waste were successfully utilized by the studied bacterial strains. Additionally, seaweed hydrolysate and digestate served as nutrient supplements supporting microbial fermentation. According to the highest achieved relative lactic acid production, a scaled-up co-fermentation of candy-waste and digestate was performed. Lactic acid reached a concentration of 65.65 g/L, with 61.69% relative lactic acid production, and 1.37 g/L/hour productivity. The findings indicate that lactic acid can be successfully produced from low-cost industrial residues.

Adaptive laboratory evolution to hypersaline conditions of lactic acid bacteria isolated from seaweed.

Seaweed biomass has been proposed as a promising alternative carbon source for fermentation processes using microbial factories. However, the high salinity content of seaweed biomass is a limiting factor in large scale fermentation processes. To address this shortcoming, three bacterial species (Pediococcus pentosaceus, Lactobacillus plantarum, and Enterococcus faecium) were isolated from seaweed biomass and evolved to increasing concentrations of NaCl. Following the evolution period, P. pentosaceus reached a plateau at the initial NaCl concentration, whereas L. plantarum, and E. faecium showed a 1.29 and 1.75-fold increase in their salt tolerance, respectively. The impact that salt evolution had on lactic acid production using hypersaline seaweed hydrolysate was investigated. Salinity evolved L. plantarum produced 1.18-fold more lactic acid than the wild type, and salinity evolved E. faecium was able to produce lactic acid, while the wild type could not. No differences in lactic acid production were observed between the P. pentosaceus salinity evolved and wild type strains. Evolved lineages were analyzed for the molecular mechanisms underlying the observed phenotypes. Mutations were observed in genes affecting the ion balance in the cell, the composition of the cell membrane and proteins acting as regulators. This study demonstrates that bacterial isolates from saline niches are promising microbial factories for the fermentation of saline substrates, without the requirement of previous desalination steps, while preserving high final product yields.

The Effect of Breastfeeding on Food Allergies in Newborns and Infants.

Breastfeeding is the preferred method of infant feeding and its establishment is one of the primary goals for the infant. Allergic diseases are common in childhood, with increased morbidity. Food allergies are also associated with a strong negative impact on health-related quality of life and is a major public health problem. In addition, maternal exclusion of common allergens during pregnancy and/or lactation suggests that supplementation with regular cow's milk formula during the first week of life should be avoided. Breast milk contains many active immune factors, such as cytokines, inflammatory mediators, signaling molecules and soluble receptors, which may also reduce the risk of allergic disease. The prophylactic effects of breastfeeding have been the subject of many studies, some with weak evidence. In this narrative review, we aim to provide an up-to-date account of the effects of prophylactic breastfeeding on food allergy and other common allergies in infants and children up to 5 years of age. Colostrum in particular has been shown to be prophylactic against food allergy. The American Academy of Pediatrics cautions that the relationship between duration of breastfeeding and incidence of food allergy in early childhood is unclear. The protective role of breastfeeding has a positive effect on allergy prevention, which is opposed by the early introduction of solid foods, but larger studies are needed to confirm the evidence. There is evidence that breastfeeding is effective in providing partial protection to infants.

CNS imaging is a key diagnostic tool in the evaluation of patients with CFC syndrome: two cases and literature review.

Cardio-facio-cutaneous (CFC) syndrome is characterized by a variable degree of cognitive impairment, and multiple congenital anomalies including characteristic facies, cardiac, and ectodermal abnormalities. CFC syndrome is caused by mutations in the genes BRAF, MEK1, or MEK2. Here we provide a follow-up report on two patients presenting distinct facial appearance and other features of the syndrome, and we present the first molecular evidence of paternal origin for a CFC-causing germline mutation. Brain imaging revealed a lipoma of the corpus callosum and periventricular leukoencephalopathy as well as a hypoplastic corpus callosum, and defects in myelinization, in each patient, respectively. A review of the literature showed that, although non-specific, ventriculomegaly, hydrocephalus, and cortical atrophy represent the most frequent imaging findings of brain anomalies in CFC syndrome. CNS abnormalities are significant diagnostic features of CFC syndrome and a brain MRI is recommended in individuals diagnosed with CFC or suspected of having CFC syndrome.

Pelvic hypoplastic kidney in a patient with Williams-Beuren syndrome.

Renal and renovascular abnormalities constitute features of the Williams-Beuren syndrome (WBS), one multisystem genetic disorder in childhood, caused by a microdeletion of chromosome 7. We report a 12 years old boy who was diagnosed with WBS and had an ectopic pelvic hypoplastic left kidney, detected by ultrasonography and renal scintigraphy. Dystopic hypoplastic kidney is an infrequent finding in patients with WBS and our report showed the importance of a complete clinical and laboratory study of renal function in WBS.

Phenotype-genotype correlation of a patient with a "balanced" translocation 9;15 and cryptic 9q34 duplication and 15q21q25 deletion.

We report on a 2-year-old boy with intellectual disabilities, distinctive facies, hypotonia, cardiac, and renal malformations. During his infancy he had recurrent episodes of apnea, cyanosis, and bradycardia. Chromosomal analysis showed a de novo apparently balanced translocation 46,XY,t(9;15)(q31;q26)dn. The use of array-comparative genomic hybridization (CGH) however, revealed the presence of additional cryptic complex chromosomal rearrangements involving a approximately 5-5.8 Mb distal duplication on chromosome 9 (9q34.1 --> 9q34.3), and deletions on three separate regions of chromosome 15 adding to approximately 8.1-12.2 Mb (15q21.2 --> 15q21.3, 15q22.31 --> 15q23, 15q25.1 --> 15q25.2). During confirmation with fluorescence in situ hybridization (FISH) an inversion was unexpectedly revealed on chromosome 15 (15q21.1 --> 15q21.2). To our knowledge this is the first patient reported whose phenotype is due to partial trisomy 9q, and complex interstitial deletions of 15q, not involving the Prader-Willi/Angelman region and encompassing the critical region 15q21q25. We provide correlation between the clinical findings of our patient and the phenotype of the 9q34 duplication syndrome, the 15q21, and the 15q25 deletion syndromes.

Case Report: Intracranial Hypertension Secondary to Guillain-Barre Syndrome.

Guillain-Barre Syndrome (GBS), a common cause of acute flaccid paralysis, is characterized by a rapidly progressive, usually symmetric weakness of the extremities. Headache and intracranial hypertension (ICHT) are very rare complications of GBS. Herein we report our current case of an obese girl with typical signs of GBS associated with autonomic dysfunction, cranial nerve deficits and increased intracranial pressure (ICP). We also perform a systematic study presenting and discussing previous case reports of GBS associated with ICHT, papilledema or hydrocephalus, highlighting the differences of the current case compared to previous studies. Although intracranial hypertension is a rare complication of pediatric GBS, clinicians should promptly detect it. Obesity may be a predisposing factor, given the strong association between idiopathic intracranial hypertension (IIH) and weight gain. Neurological evaluation, fundus examination and low threshold for intracranial imaging should be an integral part of medical practice in case of obesity, headache or visual changes in GBS patients.

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss.

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.

Cervical lymphadenopathy in childhood epidemiology and management.

Cervical lymphadenopathy (CL) is common in childhood. The aim of this study is to evaluate the etiology, follow-up, and treatment of persistent CL. The authors studied retrospectively 50 children with CL, hospitalized at the Department of Pediatrics and Pediatrics Surgery. Patients underwent ultrasonography. Thirty-six percent presented abnormal ultrasonographic image and underwent excisional biopsy. Biopsies revealed 4 thyroglossal cysts, 3 branchial cysts, 1 hemangioma, 2 sebaceous cysts, 1 dermoid cyst, 5 occurrences of tuberculosis lymphadenitis, 1 occurrence of Bartonella henselae lymphadenopathy, and 1 case of non-Hodgkin lymphoma. In conclusion, CL is usually a benign finding; bacterial and viral infections are the most common causes. Ultrasonography help in etiology and follow-up of CL.

Antenatal and postnatal evidence of periventricular leukomalacia as a further indication of vascular disruption in Adams-Oliver syndrome.

We describe a new family with Adams-Oliver syndrome (AOS). The propositus is a 14-month-old boy presenting with aplasia cutis congenita, distal limb transverse defects, growth retardation, and a wide atrial septal defect. Central nervous system abnormalities included central hypotonia, and magnetic resonance imaging (MRI) findings consistent with periventricular leukomalacia (PVL). Fetal MRI at 26 weeks' gestation had shown bilateral dilatation of lateral ventricles and periventricular cysts at the site of postnatal lesions. The patient's father and paternal grandfather also had manifestations indicative of AOS. Antenatal and postnatal MRI findings suggest that our patient's PVL represents an unusual congenital feature of AOS, possibly due to vascular disruption and decreased perfusion during critical periods of fetal brain development.

Human placental growth hormone is increased in maternal serum in pregnancies affected by Down syndrome.

OBJECTIVE: To evaluate the relationship between maternal serum levels of human placental growth hormone (hPGH) and fetal Down syndrome at gestational midtrimester. METHODS: We retrospectively analyzed samples of serum from 21 women with Down syndrome pregnancies detected at gestational midtrimester. The samples were obtained at 16-23 weeks' gestation during amniocentesis for fetal karyotyping. Sixty-two serum samples were used as controls, which were obtained at 16-23 weeks' gestation from women with singleton, uncomplicated pregnancies, who gave birth to healthy neonates with a birth weight appropriate for gestational age. The hPGH levels were measured by a solid-phase immunoradiometric assay using 2 different epitopes. RESULTS: The median hPGH values in the serum of the Down-syndrome-affected pregnancies were significantly higher (p < 0.05) than those of the normal pregnancies at 16-23 weeks' gestation: the median value in the serum was 9.4 ng/ml (5th to 95th percentiles = 1.49-39.03) versus 4.7 ng/ml (0.53-7.88). CONCLUSION: The hPGH levels in maternal serum were found to be higher at 16-23 weeks' gestation in pregnancies affected by fetal Down syndrome. Further investigation is needed to examine if maternal serum hPGH could be used as an additional marker in prenatal screening of Down syndrome at gestational midtrimester.

Stroke following Glenn anastomosis in a child with inherited thrombophilia.

The optimal anticoagulation following Fontan operation and its modifications remain controversial and it is even less well defined as regards patients with inherited thrombophilia. We present a case of a child with bidirectional Glenn anastomosis for double inlet left ventricle that suffered a stroke despite aspirin prophylaxis; the patient was combined homozygous for prothrombin G20210A mutation and for methylenetetrahydrofolate reductase C677T mutation as well. The family history was positive for fetal loss and premature cardiovascular disease. Large-scale studies are needed to evaluate whether carriers of thrombophilia mutations need more intense thromboprophylaxis.

3C syndrome with cryptorchidism and posterior embryotoxon.

We report a case of the 3C (cranio-cerebello-cardiac) syndrome, also known as Ritscher-Schinzel syndrome, a rare autosomal recessive disorder characterized by craniofacial, cerebellar, and cardiac anomalies. In addition to features previously reported the child had Wormian bones of the skull, intra-abdominal testes, and posterior embryotoxon that have not previously been reported as part of the 3C syndrome.

Can low doses of simvastatin enhance fracture healing? An experimental study in rabbits.

Several observational and experimental studies have investigated the potential anabolic effects of statins on undisturbed bone but only a few recent studies have examined the effect of statins on skeletal repair. The goal of the study is to investigate any potential early anabolic effect of the systemic administration of simvastatin in low doses (based on earlier safety and efficacy studies on undisturbed bone) on fracture healing. Fifty-four skeletally mature male New Zealand White rabbits were used for the study. The rabbits were assigned to one of three experimental groups: a control group, and two groups that were orally administrated a diet with 10 and 30 mg/kg/day of simvastatin, respectively. A complete biochemical blood count was performed to exclude drug-induced complications. Half of the animals of each group were sacrificed at 15 days and the other half at 30 days after surgery at which time intervals healing quality was assessed. The bones were subjected to biomechanical testing, histomorphometric analysis and peripheral quantitative computed tomography. In animals received simvastatin of 30 mg/kg/day a significant reduction of BMD, stiffness, and energy absorbed to failure were observed. At 15 days, the amount of cartilaginous callus formation was reduced, and the void space was significantly increased, in the animals of both groups that received simvastatin when compared to the control group (p<.05). Our results suggest that simvastatin doses of 30 mg/kg/day may have a negative anabolic effect on callus formation in rabbits, whereas doses of 10 mg/kg/day seem not to produce a significant positive or a negative effect, especially at the early stages of fracture remodeling.

Cryptic genomic imbalances in patients with de novo or familial apparently balanced translocations and abnormal phenotype.

BACKGROUND: Carriers of apparently balanced translocations are usually phenotypically normal; however in about 6% of de novo cases, an abnormal phenotype is present. In the current study we investigated 12 patients, six de novo and six familial, with apparently balanced translocations and mental retardation and/or congenital malformations by applying 1 Mb resolution array-CGH. In all de novo cases, only the patient was a carrier of the translocation and had abnormal phenotype. In five out of the six familial cases, the phenotype of the patient was abnormal, although the karyotype appeared identical to other phenotypically normal carriers of the family. In the sixth familial case, all carriers of the translocations had an abnormal phenotype. RESULTS: Chromosomal and FISH analyses suggested that the rearrangements were "truly balanced" in all patients. However, array-CGH, revealed cryptic imbalances in three cases (3/12, 25%), two de novo (2/12, 33.3%) and one familial (1/12, 16.6%). The nature and type of abnormalities differed among the cases. In the first case, what was identified as a de novo t(9;15)(q31;q26.1), a complex rearrangement was revealed involving a ~6.1 Mb duplication on the long arm of chromosome 9, an ~10 Mb deletion and an inversion both on the long arm of chromosome 15. These imbalances were located near the translocation breakpoints. In the second case of a de novo t(4;9)(q25;q21.2), an ~6.6 Mb deletion was identified on the short arm of chromosome 7 which is unrelated to the translocation. In the third case, of a familial, t(4;7)(q13.3;p15.3), two deletions of ~4.3 Mb and ~2.3 Mb were found, each at one of the two translocation breakpoints. In the remaining cases the translocations appeared balanced at 1 Mb resolution. CONCLUSION: This study investigated both de novo and familial apparently balanced translocations unlike other relatively large studies which are mainly focused on de novo cases. This study provides additional evidence that cryptic genomic imbalances are common in patients with abnormal phenotype and "apparently balanced" translocations not only in de novo but can also occur in familial cases. The use of microarrays with higher resolution such as oligo-arrays may reveal that the frequency of cryptic genomic imbalances among these patients is higher.