Development of a high-resolution typing method for SLA-3, swine MHC class I antigen 3.

We developed a high-resolution and comprehensive typing method for swine leukocyte antigen 3 (SLA-3), an MHC class I gene, employing locus-specific genomic PCR followed by subsequent direct sequencing. A total of 292 individuals from nine pure, one cross-breed and six cell lines were successfully typed. A total of 21 SLA-3 alleles were identified, of which four were found to be novel alleles. However, the allelic diversity of SLA-3 was lower than that of previously reported class I genes, SLA-1 and -2. More SLA-3 alleles were observed in the Landrace and Yorkshire breeds than the other breeds. SLA-3*04:01 was identified in seven out of nine breeds and was the most widely distributed allele across all breeds. Therefore, the typing method reported in this study completes our efforts to develop high-resolution typing methods for major SLA molecules, facilitating the combined analysis of major SLA genes from field samples, which is important to understand the relationship between the adaptive immune responses against pathogens and the immunogenetic makeup of an individual.

Identification of a novel HLA-A*02 allele, HLA-A*02:01:01:32, in a deceased Caucasoid donor.

HLA-A*02:01:01:32 differs from HLA-A*02:01:01:01 by a single nucleotide substitution (G→A) at position 2456.

The major histocompatibility complex homozygous inbred Babraham pig as a resource for veterinary and translational medicine.

The Babraham pig is a highly inbred breed first developed in the United Kingdom approximately 50 years ago. Previous reports indicate a very high degree of homozygosity across the genome, including the major histocompatibility complex (MHC) region, but confirmation of homozygosity at the specific MHC loci was lacking. Using both direct sequencing and PCR-based sequence-specific typing, we confirm that Babraham pigs are essentially homozygous at their MHC loci and formalise their MHC haplotype as Hp-55.6. This enhances the utility of the Babraham pig as a useful biomedical model for studies in which controlling for genetic variation is important.

Paradox of serial interferon-gamma release assays: variability width more important than specificity size.

SETTING: Serial screening for latent tuberculous infection (LTBI) is commonly performed in certain populations, such as health care workers. The high apparent conversion rate in some studies of interferon-gamma release assays is puzzling given the claimed high specificity of these tests. OBJECTIVE: To understand how test-retest variability, specificity, and underlying LTBI prevalence affect observed outcomes of repeated testing for LTBI. DESIGN: Mathematical model assuming constant test sensitivity and specificity over time and no new infections. RESULTS: Test-retest variability had a large effect on the observed proportion of conversions (initial negative test, followed by a positive test) and reversions (initial positive test, followed by a negative test). For example, a test with 70% specificity and 5% test-retest variability would be associated with a conversion rate of 3.7% and a reversion rate of 7.7%, while a test with 95% specificity but 10% test-retest variability would be associated with a conversion rate of 5.5% and a reversion rate of 57%, assuming that both tests are 80% sensitive and underlying LTBI prevalence was 5%. CONCLUSION: Test-retest variability is a key parameter that should be reported for tests used for serial screening for LTBI. Reducing test-retest variability can reduce false-positive and false-negative results.

Intraspinal schwannoma and neurogenic bladder.

Most lumbar intradural schwannomas present initially as radiculopathies with sensory disturbances. However, neurogenic bladder dysfunction may be one of the earliest manifestations and can cause long-term disability. We present the case of a patient with a L3-4 schwannoma (newly diagnosed owing to recurrent urinary retention and urinary tract infection) who finally underwent surgical resection. Improvement of bladder sensation was documented by urodynamic study and the patient was subsequently weaned off her Foley catheter with satisfactory outcome.

Beating 'Guangdong cancer': a review and update on nasopharyngeal cancer.

Once endemic in southern China, nasopharyngeal cancer is becoming less prevalent in Hong Kong. This is probably due to a better understanding of the risk factors associated with the disease, its genomic landscape, advances in radiotherapy technology, and development of effective systemic agents. More specifically, the close relationship between Epstein-Barr virus and nasopharyngeal cancer opens up the possibility of using Epstein-Barr virus DNA as a biomarker for early detection and monitoring of the disease. On the other hand, the looming genomic data for nasopharyngeal cancer aid in the development of powerful biomarkers and promising targeted therapy. Clinical use of a combination of radiotherapy and chemotherapy continues to increase, while the development of immunotherapy, such as checkpoint inhibitors, offers hope in improving treatment outcome.

A randomized placebo controlled trial of vitamin B12 supplementation to prevent cognitive decline in older diabetic people with borderline low serum vitamin B12.

BACKGROUND & AIMS: Older diabetic people are at risk of cognitive decline. Vitamin B12 deficiency in older people is associated with cognitive impairment and Alzheimer's disease. Vitamin B12 deficiency may therefore contribute to cognitive decline in older diabetic people. We therefore performed a randomized placebo-controlled trial of vitamin B12 supplementation to prevent cognitive decline in older diabetic people with mild vitamin B12 deficiency. METHODS: 271 diabetic non-demented outpatients aged 70 years or older with plasma vitamin B12 150-300 pmol/L in outpatient clinics were randomly assigned to take either methylcobalamin 1000 µg or two similar looking placebo tablets once daily for 27 months. All subjects were followed up at 9 monthly intervals. The primary outcome is cognitive decline as defined by an increase in clinical dementia rating scale (CDR) global score. The secondary outcomes included Neuropsychological Test Battery (NTB) z-scores, serum methymalonic acid (MMA) and homocysteine. RESULTS: The subjects in the trial groups were well matched in clinical characteristics, except that active intervention group had more smokers. 46.5% and 74.1% had elevated serum methymalonic acid (≥0.21 µmol/L) and homocysteine (≥13 µmol/L) respectively. 44% of the subjects had CDR score of 0.5 suggesting questionable dementia. At month 9 and 27, serum MMA and homocysteine was significantly reduced in the active treatment group, when compared with placebo group. (P < 0.0001, student t test) At month 27, there was no significant group difference in changes in CDR or NTB z-scores. Exclusion of smokers did not alter the results. Subgroup analysis of high MMSE and serum MMA showed similar results. CONCLUSION: Vitamin B12 supplementation did not prevent cognitive decline in older diabetic patients with borderline vitamin B12 status. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT02457507.

Prevalence of vitamin D insufficiency among adolescents and its correlation with bone parameters using high-resolution peripheral quantitative computed tomography.

UNLABELLED: Vitamin D deficiency and insufficiency are highly prevalent among adolescents in Hong Kong, which is a sub-tropical city with ample sunshine. Vitamin D level is significantly correlated with key bone density and bone quality parameters. Further interventional studies are warranted to define the role of vitamin D supplementation for improvement of bone health among adolescents. INTRODUCTION: The relationship between bone quality parameters and vitamin D (Vit-D) status remains undefined among adolescents. The aims of this study were to evaluate Vit-D status and its association with both bone density and bone quality parameters among adolescents. METHODS: Three hundred thirty-three girls and 230 boys (12-16 years old) with normal health were recruited in summer and winter separately from local schools. Serum 25(OH) Vit-D level, bone density and quality parameters by Dual Energy X-ray Absorptiometry (DXA) and High-Resolution peripheral Quantitative Computed Tomography (HR-pQCT), dietary calcium intake, and physical activity level were assessed. RESULTS: Sixty-four point seven percent and 11.4 % of subjects were insufficient [25 ≤ 25(OH)Vit-D ≤ 50 nmol/L] and deficient [25(OH)Vit-D < 25 nmol/L] in Vit-D, respectively. The mean level of serum 25(OH)Vit-D in summer was significantly higher than that in winter (44.7 ± 13.6 and 35.9 ± 12.6 nmol/L, respectively) without obvious gender difference. In girls, areal bone mineral density (aBMD) and bone mineral content (BMC) of bilateral femoral necks, cortical area, cortical thickness, total volumetric bone mineral density (vBMD), and trabecular thickness were significantly correlated with 25(OH)Vit-D levels. In boys, aBMD of bilateral femoral necks, BMC of the dominant femoral neck, cortical area, cortical thickness, total vBMD, trabecular vBMD, BV/TV, and trabecular separation were significantly correlated with 25(OH)Vit-D levels. CONCLUSION: Vit-D insufficiency was highly prevalent among adolescents in Hong Kong with significant correlation between Vit-D levels and key bone density and bone quality parameters being detected in this study. Given that this is a cross-sectional study and causality relationship cannot be inferred, further interventional studies investigating the role of Vit-D supplementation on improving bone health among adolescents are warranted.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Sequence variations of the locus-specific 5' untranslated regions of SLA class I genes and the development of a comprehensive genomic DNA-based high-resolution typing method for SLA-2.

The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs.

Can soy intake affect serum uric acid level? Pooled analysis from two 6-month randomized controlled trials among Chinese postmenopausal women with prediabetes or prehypertension.

PURPOSE: Hyperuricemia is a recognized risk factor for cardiovascular diseases. Soy foods contain a moderate amount of purine and may predispose to raised serum uric acid (UA). However, no study has examined the long-term effect of soy intake on UA levels. We examined whether consumption of soy foods and isoflavone extracts for 6 months altered serum UA. METHODS: The analysis included two randomized controlled trials (soy protein trial and whole soy trial) among total 450 postmenopausal women with either prehypertension or prediabetes. We conducted a pooled analysis by combining participants from both the soy flour and soy protein groups (combined soy foods group), participants from both the isoflavone and daidzein groups (combined isoflavone group) and participants from both milk placebo groups. Fasting venous samples were obtained at baseline and the end of the trial for serum UA analysis. RESULTS: In the pooled data, 417 subjects completed the study according to protocol. The baseline serum UA levels were comparable among the three combined groups. There was a lower decrease in UA levels among women in the combined soy foods group compared with women in the other two groups (p = 0.028 and 0.026). The net decrease and % decrease in UA were 14.5 µmol/L (95 % CI 1.93-25.6, p = 0.023) or 4.9 % (95 % CI 1.3-8.5 %, p = 0.023) between the combined soy foods group and placebo group. CONCLUSIONS: Among Chinese postmenopausal women with either prehypertension or prediabetes, soy intake did not increase urate levels.

Sequence-based characterization of five SLA loci in Asian wild boars.

Two swine leucocyte antigen (SLA) class I (SLA-1 and SLA-2) and three class II (DRB1, DQB1 and DQA) genes were investigated for their diversity in Asian wild boars using a sequence-based typing method. A total of 15 alleles were detected at these loci, with eleven being novel. The findings provide one of the first glimpses of the SLA allelic diversity and architecture in the wild boar populations.

Ge overlayer and surface alloy structures on Pt(100) studied using alkali ion scattering spectroscopy, x-ray photoelectron spectroscopy and x-ray photoelectron diffraction.

We have investigated surface structures formed by deposition of Ge on a Pt(100) substrate by using a multi-technique approach utilizing alkali ion scattering spectroscopy (ALISS), x-ray photoelectron spectroscopy (XPS), and x-ray photoelectron diffraction (XPD). ALISS was used to distinguish Ge overlayers from incorporated alloy layers for the surface structures reported, and to supply structural information about the surface alloy or 'layer compound' formed by the deposition of 1.5-ML Ge. A Ge adlayer forms following the deposition of 0.2-ML Ge on Pt(100) and annealing at 600 K. ALISS revealed that Ge adatoms in these overlayers had 1D (incomplete c(2 × 2)) Ge-Ge ordering along [010] and equivalent directions, even though this was not directly apparent in observations using LEED and STM. A c(2 × 2)-Ge overlayer was produced after 0.5 ML-Ge deposition on Pt(100) and annealing at 600 K. Deposition of 1.5-ML Ge on Pt(100) and annealing at 600 K caused extensive Ge interdiffusion into the third (subsurface) layer, while the first and second layers remained as a c(2 × 2) Ge overlayer and (1 × 1) Pt layer, respectively. We propose that the Pt(100) substrate thus is 'capped' by an alloy film with the structure of a body-centered tetragonal Pt2Ge layer compound, which is terminated by a pure-Ge layer that is indistinguishable from a c(2 × 2)-Ge adlayer. This explains the apparently 'strange' result that even though extensive Ge interdiffusion was occurring deeply into the Pt bulk during annealing at 900 and 1200 K, a Ge overlayer remained on the surface. XPS spectra showed a +0.5 eV binding energy shift of the Ge 3d core level and a small (0-0.1 eV) positive shift of the Pt 5d core level compared to Ge(100) and Pt(100) surfaces for the c(2 × 2)-Ge overlayer. There was no effect on these binding energies upon formation of the Pt2Ge layer compound at the surface, and this indicates similar Ge-Pt interactions in the two cases. Compared to other overlayers of Group-IV atoms on metal surfaces, the Ge overlayer on Pt(100) was extraordinarily stable.

Development and validation of a sensitive method for simultaneous determination of rosuvastatin and N-desmethyl rosuvastatin in human plasma using liquid chromatography/negative electrospray ionization/tandem mass spectrometry.

A sensitive and specific liquid chromatography tandem mass spectrometric method was developed and validated for the simultaneous determination of rosuvastatin (ROS) and N-desmethyl rosuvastatin (NOR-ROS) in human plasma using deuterium-labeled internal standards. The plasma samples were prepared using liquid-liquid extraction with diethyl ether. Chromatographic separation was accomplished on an Xterra MS C18 column. The mobile phase consisted of a gradient mixture of 15 µmol/L ammonium acetate in water and in methanol, maintained at a flow rate of 0.4 mL/min. Mass spectrometric detection was carried out in negative electrospray ionization mode and monitored by quantification and qualification transitions for each analyte. Using 300 µL plasma samples, the lower limits of quantification of ROS and NOR-ROS were 0.05 and 0.02 µg/L respectively. The linearity of ROS and NOR-ROS ranged from 0.05 to 42 and 0.02 to 14 µg/L respectively. The relative standard deviations of ROS and NOR-ROS were <13 and 9%, respectively, while the deviations from expected values were within -4.7-9.8 and -5.2-4.6%, respectively. The present method offered high sensitivity and was successfully applied to a 24 h pharmacokinetic study of ROS and NOR-ROS in healthy subjects receiving a single dose of 10 mg ROS.

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

Associations of uric acid and gamma-glutamyltransferase (GGT) with obesity and components of metabolic syndrome in children and adolescents.

BACKGROUND: The combined effect of uric acid, gamma-glutamyltransferase (GGT) and cardiovascular risk factors clustering in the youth remains under-explored. OBJECTIVE: The objective of this study was to examine the association between uric acid, GGT, obesity and the individual components of metabolic syndrome in children and adolescents. METHODS: We performed a cross-sectional observational study of 2067 children and adolescents (875 boys and 1192 girls) aged 6-20 years who were healthy volunteers and were recruited from primary and secondary schools in Hong Kong between 2007 and 2008. Subjects were divided into two strata (75th percentile as cut-off) for comparison between odds of cardiovascular risk factors. RESULTS: After adjustment by multivariable logistic regression, subjects in upper stratum, i.e., >75th percentile, of either serum uric acid or GGT levels were associated with obesity, low high-density lipoprotein cholesterol (HDL-C) level and high blood pressure (adjusted odds ratios [AOR] ranged from 1.63 to 5.82, all P < 0.005) compared with those in the lower stratum. There were combined effect for upper stratum of both uric acid and GGT in the association with obesity, low HDL-C and high blood pressure (AOR ranged from 2.60 to 10.69, all P < 0.05) after adjustment for age, sex and body mass index z-score (except for obesity status) as well as body height (for high blood pressure). CONCLUSION: Uric acid and GGT have combined effect in association with obesity and other cardiovascular risk factors in children and adolescents.

HAMLET: functional properties and therapeutic potential.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.