Expanding the clinical and mutational spectrum of Kaufman oculocerebrofacial syndrome with biallelic UBE3B mutations

Biallelic mutations of UBE3B have recently been shown to cause Kaufman oculocerebrofacial syndrome (also reported as blepharophimosis–ptosis–intellectual disability syndrome), an autosomal recessive condition characterized by hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels. To date, six patients with either missense mutations affecting the UBE3B HECT domain or truncating mutations have been described. Here, we report on the identification of homozygous or compound heterozygous UBE3B mutations in six additional patients from five unrelated families using either targeted UBE3B sequencing in individuals with suggestive facial dysmorphic features, or exome sequencing. Our results expand the clinical and mutational spectrum of the UBE3B-related disorder in several ways. First, we have identified UBE3B mutations in individuals who previously received distinct clinical diagnoses: two sibs with Toriello–Carey syndrome as well as the patient reported to have a “new” syndrome by Buntinx and Majewski in 1990. Second, we describe the adult phenotype and clinical variability of the syndrome. Third, we report on the first instance of homozygous missense alterations outside the HECT domain of UBE3B, observed in a patient with mildly dysmorphic facial features. We conclude that UBE3B mutations cause a clinically recognizable and possibly underdiagnosed syndrome characterized by distinct craniofacial features, hypotonia, failure to thrive, eye abnormalities, other congenital malformations, low cholesterol levels, and severe intellectual disability. We review the UBE3B-associated phenotypes, including forms that can mimick Toriello–Carey syndrome, and suggest the single designation “Kaufman oculocerebrofacial syndrome”.     

First Detection of Leishmania tropica DNA and Trypanosoma Species in Sergentomyia Sand Flies (Diptera: Psychodidae) from an Outbreak Area of Cutaneous Leishmaniasis in Ghana

Background

Leishmania major and an uncharacterized species have been reported from human patients in a cutaneous leishmaniasis (CL) outbreak area in Ghana. Reports from the area indicate the presence of anthropophilic Sergentomyia species that were found with Leishmania DNA.

Methodology/Principal Findings

In this study, we analyzed the Leishmania DNA positive sand fly pools by PCR-RFLP and ITS1 gene sequencing. The trypanosome was determined using the SSU rRNA gene sequence. We observed DNA of L. major, L. tropica and Trypanosoma species to be associated with the sand fly infections. This study provides the first detection of L. tropica DNA and Trypanosoma species as well as the confirmation of L. major DNA within Sergentomyia sand flies in Ghana and suggests that S. ingrami and S. hamoni are possible vectors of CL in the study area.

Conclusions/Significance

The detection of L. tropica DNA in this CL focus is a novel finding in Ghana as well as West Africa. In addition, the unexpected infection of Trypanosoma DNA within S. africana africana indicates that more attention is necessary when identifying parasitic organisms by PCR within sand fly vectors in Ghana and other areas where leishmaniasis is endemic.     

Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.Key words: monoclonal antibody, FcRn, binding affinity, subcutaneous bioavailability, semi-mechanism-based pharmacokinetic model     

β-Cells with relative low HIMP1 overexpression levels in a transgenic mouse line enhance basal insulin production and hypoxia/hypoglycemia tolerance

Rodent pancreatic β-cells that naturally lack hypoglycemia/hypoxia inducible mitochondrial protein 1 (HIMP1) are susceptible to hypoglycemia and hypoxia influences. A linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 is suggested in a recent study using transformed β-cells lines. To further illuminate this linkage, we applied mouse insulin 1 gene promoter (MIP) to control HIMP1-a isoform cDNA and have generated three lines (L1 to L3) of heterozygous HIMP1 transgenic (Tg) mice by breeding of three founders with C57BL/6J mice. In HIMP1-Tg mice/islets, we performed quantitative polymerase chain reaction (PCR), immunoblot, histology, and physiology studies to investigate HIMP1 overexpression and its link to β-cell function/survival and body glucose homeostasis. We found that the HIMP1 level increased steadily in β-cells of L1 to L3 heterozygous HIMP1-Tg mice. HIMP1 overexpression at relatively lower levels in L1 heterozygotes results in a negligible decline in blood glucose concentrations and an insignificant elevation in blood insulin levels, while HIMP1 overexpression at higher levels are toxic, causing hyperglycemia in L2/3 heterozygotes. Follow-up studies in 5-30-week-old L1 heterozygous mice/islets found that HIMP1 overexpression at relatively lower levels in β-cells has enhanced basal insulin biosynthesis, basal insulin secretion, and tolerances to low oxygen/glucose influences. The findings enforced the linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 in β-cells, and show a potential value of HIMP1 overexpression at relatively lower levels in modulating β-cell function and survival.     

A combination of stomata deregulation and a distinctive modulation of amino acid metabolism are associated with enhanced tolerance of wheat varieties to transient drought

Introduction

Mediterranean winter crops are commonly and increasingly exposed to irregular rainfall and high temperatures, which lead to transient drought events of different degrees, adversely affecting growth and yield. Hence, exploring the diverse degrees of tolerance to drought existing in the crop and the molecular strategies behind it is pivotal for the development of ad hoc breeding programs.

Objective

We investigated the physiological and metabolic response of six commercial wheat cultivars to transient water stress at the tillering and grain-filling stages.

Methods

Drought experiments in lysimeters were set up at two developmental stages including six wheat cultivars. Newly expanded youngest leaves and flag leaves were sampled during the drought and following recovery. Metabolite profiles were generated using a GC–MS based protocol. Data on transpiration were continually acquired by measuring the weight variation of pots using electronic temperature compensated load cells.

Results

The tillering stage in wheat is more sensitive to droughts than the grain filling stage. The former stage was characterized by pronounced metabolic alterations also during recovery from the drought, and plants exhibited reduced transpiration. Notably, cultivars varied considerably in their susceptibility to drought. Exceptionally only in cv Zahir was transpiration not reduced at tillering. During recovery, the transpiration rate of Yuval and Zahir was not significantly affected, while except Ruta the other varieties maintained lower values. At grain-filling, a moderate decrease in transpiration in response to drought was evident in Bar-Nir, Yuval and Zahir varieties as compared with the stronger response of Gedera, Galil and Ruta. The transpiration trend during recovery remained lower than the control plants, particularly in Gedera and Zahir, while it reached higher values than control plants in Yuval and Ruta varieties. Metabolite profiling of leaves across cultivars showed varietal specific trends of response. Particularly during tillering, amino acid metabolism was differentially regulated across cultivars. For instance, Ruta and Zahir exhibited major changes in central carbon nitrogen metabolism during stress response, accumulating large amounts of proline and threonine during tillering, while in Bar-Nir a general decrease in relative amino acid content was noted. Changes in stress related GABA were common to Galil, Ruta, Yuval and Zahir. Desiccation related raffinose family oligosaccharides were mostly associated with a later stage of grain-filling and recovery stages of response.

Conclusion

The results indicate the occurrence of stage-dependent metabolic diversification along with a physiological response during transient droughts among wheat cultivars. It can be concluded that the most tolerant cultivar was Zahir, where a combination of stomatal closure deregulation and a significant accumulation rate of stress-related metabolites were evident.

     

A bell pepper cultivar tolerant to chilling enhanced nitrogen allocation and stress‐related metabolite accumulation in the roots in response to low root‐zone temperature

Two bell pepper (Capsicum annuum) cultivars, differing in their response to chilling, were exposed to three levels of root‐zone temperatures. Gas exchange, shoot and root phenology, and the pattern of change of the central metabolites and secondary metabolites caffeate and benzoate in the leaves and roots were profiled. Low root‐zone temperature significantly inhibited gaseous exchange, with a greater effect on the sensitive commercial pepper hybrid (Canon) than on the new hybrid bred to enhance abiotic stress tolerance (S103). The latter was less affected by the treatment with respect to plant height, shoot dry mass, root maximum length, root projected area, number of root tips and root dry mass. More carbon was allocated to the leaves of S103 than nitrogen at 17°C, while in the roots at 17°C, more nitrogen was allocated and the ratio between C/N decreased. Metabolite profiling showed greater increase in the root than in the leaves. Leaf response between the two cultivars differed significantly. The roots accumulated stress‐related metabolites including γ‐aminobutyric acid (GABA), proline, galactinol and raffinose and at chilling (7°C) resulted in an increase of sugars in both cultivars. Our results suggest that the enhanced tolerance of S103 to root cold stress, reflected in the relative maintenance of shoot and root growth, is likely linked to a more effective regulation of photosynthesis facilitated by the induction of stress‐related metabolism.     

Cerebro-Oculo-Facio-Skeletal Syndrome with a Nucleotide Excision–Repair Defect and a Mutated XPD Gene, with Prenatal Diagnosis in a Triplet Pregnancy

Cerebro-oculo-facio-skeletal (COFS) syndrome is a recessively inherited rapidly progressive neurologic disorder leading to brain atrophy, with calcifications, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low to normal birth weight, growth failure, brain dysmyelination with calcium deposits, cutaneous photosensitivity, pigmentary retinopathy and/or cataracts, and sensorineural hearing loss. Cultured CS cells are hypersensitive to UV radiation, because of impaired nucleotide-excision repair (NER) of UV-induced damage in actively transcribed DNA, whereas global genome NER is unaffected. The abnormalities in CS are caused by mutated CSA or CSB genes. Another class of patients with CS symptoms have mutations in the XPB, XPD, or XPG genes, which result in UV hypersensitivity as well as defective global NER; such patients may concurrently have clinical features of another NER syndrome, xeroderma pigmentosum (XP). Clinically observed similarities between COFS syndrome and CS have been followed by discoveries of cases of COFS syndrome that are associated with mutations in the XPG and CSB genes. Here we report the first involvement of the XPD gene in a new case of UV-sensitive COFS syndrome, with heterozygous substitutions-a R616W null mutation (previously seen in patients in XP complementation group D) and a unique D681N mutation-demonstrating that a third gene can be involved in COFS syndrome. We propose that COFS syndrome be included within the already known spectrum of NER disorders: XP, CS, and trichothiodystrophy. We predict that future patients with COFS syndrome will be found to have mutations in the CSA or XPB genes, and we document successful use of DNA repair for prenatal diagnosis in triplet and singleton pregnancies at risk for COFS syndrome. This result strongly underlines the need for screening of patients with COFS syndrome, for either UV sensitivity or DNA-repair abnormalities.     

Antigenic glycans in parasitic infections: implications for vaccines and diagnostics

Infections by parasitic protozoans and helminths are a major world-wide health concern, but no vaccines exist to the major human parasitic diseases, such as malaria, African trypanosomiasis, amebiasis, leishmaniasis, schistosomiasis, and lymphatic filariasis. Recent studies on a number of parasites indicate that immune responses to parasites in infected animals and humans are directed to glycan determinants within cell surface and secreted glycoconjugates and that glycoconjugates are important in host-parasite interactions. Because of the tremendous success achieved recently in generating carbohydrate-protein conjugate vaccines toward microbial infections, such as Haemophilus influenzae type b, there is renewed interest in defining parasite-derived glycans in the prospect of developing conjugate vaccines and new diagnostics for parasitic infections. Parasite-derived glycans are compelling vaccine targets because they have structural features that distinguish them from mammalian glycans. There have been exciting new developments in techniques for glycan analysis and the methods for synthesizing oligosaccharides by chemical or combined chemo-enzymatic approaches that now make it feasible to generate parasite glycans to test as vaccine candidates. Here, we highlight recent progress made in elucidating the immunogenicity of glycans from some of the major human and animal parasites, the potential for developing conjugate vaccines for parasitic infections, and the possible utilization of these novel glycans in diagnostics.     

Down‐regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L^?1, 50 μL), the combination with metformin (60 mmol L^?1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.