Convolutional Neural Networks Enable Highly Accurate and Automated Subvisible Particulate Classification of Biopharmaceuticals.

Quantification of subvisible particles, which are generally defined as those ranging in size from 2 to 100 µm, is important as critical characteristics for biopharmaceutical formulation development. Micro Flow Imaging (MFI) provides quantifiable morphological parameters to study both the size and type of subvisible particles, including proteinaceous particles as well as non-proteinaceous features incl. silicone oil droplets, air bubble droplets, etc., thus enabling quantitative and categorical particle attribute reporting for quality control. However, limitations in routine MFI image analysis can hinder accurate subvisible particle classification. In this work, we custom-built a subvisible particle-aware Convolutional Neural Network, SVNet, which has a very small computational footprint, and achieves comparable performance to prior state-of-art image classification models. SVNet significantly improves upon current standard operating procedures for subvisible particulate assessments as confirmed by thorough real-world validation studies.

Wald tests for variance-adjusted equivalence assessment with normal endpoints.

Equivalence tests may be tested with mean difference against a margin adjusted for variance. The justification of using variance adjusted non-inferiority or equivalence margin is for the consideration that a larger margin should be used with large measurement variability. However, under the null hypothesis, the test statistic does not follow a t-distribution or any well-known distribution even when the measurement is normally distributed. In this study, we investigate asymptotic tests for testing the equivalence hypothesis. We apply the Wald test statistic and construct three Wald tests that differ in their estimates of variances. These estimates of variances include the maximum likelihood estimate (MLE), the uniformly minimum variance unbiased estimate (UMVUE), and the constrained maximum likelihood estimate (CMLE). We evaluate the performance of these three tests in terms of type I error rate control and power using simulations under a variety of settings. Our empirical results show that the asymptotic normalized tests are conservative in most settings, while the Wald tests based on ML- and UMVU-method could produce inflated significance levels when group sizes are unequal. However, the Wald test based on CML-method provides an improvement in power over the other two Wald tests for medium and small sample size studies.

Testing for polygenic effects in genome-wide association studies.

To confirm associations with a large number of single nucleotide polymorphisms (SNPs), each with only a small effect size, as hypothesized in the polygenic theory for schizophrenia, the International Schizophrenia Consortium (2009, Nature 460:748-752) proposed a polygenic risk score (PRS) test and demonstrated its effectiveness when applied to psychiatric disorders. The basic idea of the PRS test is to use a half of the sample to select and up-weight those more likely to be associated SNPs, and then use the other half of the sample to test for aggregated effects of the selected SNPs. Intrigued by the novelty and increasing use of the PRS test, we aimed to evaluate and improve its performance for GWAS data. First, by an analysis of the PRS test, we point out its connection with the Sum test [Chapman and Whittaker, Genet Epidemiol 32:560-566; Pan, Genet Epidemiol 33:497-507]; given the known advantages and disadvantages of the Sum test, this connection motivated the development of several other polygenic tests, some of which may be more powerful than the PRS test under certain situations. Second, more importantly, to overcome the low statistical efficiency of the data-splitting strategy as adopted in the PRS test, we reformulate and thus modify the PRS test, obtaining several adaptive tests, which are closely related to the adaptive sum of powered score (SPU) test studied in the context of rare variant analysis [Pan et al., 2014, Genetics 197:1081-1095]. We use both simulated data and a real GWAS dataset of alcohol dependence to show dramatically improved power of the new tests over the PRS test; due to its superior performance and simplicity, we recommend the whole sample-based adaptive SPU test for polygenic testing. We hope to raise the awareness of the limitations of the PRS test and potential power gain of the adaptive SPU test.

Establishing consistency across all regions in a multi-regional clinical trial.

In recent years, global collaboration has become a conventional strategy for new drug development. To accelerate the development process and shorten approval time, the design of multi-regional clinical trials (MRCTs) incorporates subjects from many countries/regions around the world under the same protocol. After showing the overall efficacy of a drug in a global trial, one can also simultaneously evaluate the possibility of applying the overall trial results to all regions and subsequently support drug registration in each region. However, most of the recent approaches developed for the design and evaluation of MRCTs focus on establishing criteria to examine whether the overall results from the MRCT can be applied to a specific region. In this paper, we use the consistency criterion of Method 1 from the Japanese Ministry of Health, Labour and Welfare (MHLW) guidance to assess whether the overall results from the MRCT can be applied to all regions. Sample size determination for the MRCT is also provided to take all the consistency criteria from each individual region into account. Numerical examples are given to illustrate applications of the proposed approach.

Adsorption of Cu(II) from aqueous solution by wine processing waste sludge.

Wine processing waste sludge (WPWS) has been shown to be an effective sorbent for sorption of nickel, lead, and chromium, but the sorption of copper (Cu) in aqueous solution by WPWS has not been conducted. The objective of this study was to explore the sorption mechanism of WPWS for copper. Infrared analysis revealed carboxyl was the major functional group in WPWS. The WPWS sorption isotherms of copper were only well described by Langmuir sorption isotherm. The maximum adsorption capacity (Qm) was 14.26 mg/g at 50 degrees C. A pseudo-second-order sorption kinetic model successfully described the kinetics of copper sorption onto WPWS. The Gibb free energies (deltaG0) ranged from -20.69 to -24.29 kJ mol(-1), and the deltaH0 and deltaS0 were 5.048 kJ mol(-1) and 91.05 J mol(-1) K(-1), respectively. The trend of the intra-particular diffusion rate is the opposite of the adsorption constant of the pseudo-second-order equation.

Removal of methylene blue from aqueous solution using wine-processing waste sludge.

Dye wastewaters usually contain toxins and high chroma, making them difficult to treat with biological methods. The adsorption process plays an important role in removing dyes from wastewaters. This study aimed to explore the methylene blue (MB) adsorption mechanism by wine-processing waste sludge (WPWS). The WPWS contains a high cation-exchange capacity (64.2 cmol(c) kg(-1)) and organic matter (52.8%). The parameters affecting MB adsorption included pH, initial concentration of MB, reaction temperature, particle size and dosage of WPWS. The WPWS adsorption isotherms of MB were only well described by Langmuir adsorption isotherm. The maximum adsorption capacity (Q(m)) of MB was 285.7 mg g(-1) at 25 °C. The activation energy determined by Arrhenius equation is 29.995 kJ mol(-1). Under steady-state reaction conditions, the Gibb free energy (ΔG°) ranged from -24.607 to -27.092 kJ mol(-1) and ΔH° was -8.926 kJ mol(-1), indicating that lower reaction temperature would favor MB adsorption. Therefore, MB adsorption by WPWS was a spontaneous, exothermic and physisorption reaction.

Autogenous iliotibial band enhancement combined with tendon lengthening plasty to treat patella baja: A case report.

BACKGROUND: Patella baja is a severe complication after knee injury or surgery, resulting in pain and impaired movement. This disorder is also a substantial challenge for orthopaedic surgeons. Currently, no consensus exists regarding the gold standard management of patella baja. If not appropriately treated, significant dysfunction of the knee joint will occur. CASE SUMMARY: A 46-year-old man with a left patellar fracture was treated with tension band fixation at a local hospital. He had undergone a second operation at the same hospital because of limited knee flexion 6 mo after surgery. Unfortunately, the patellar tendon was ruptured. The patellar tendon was subsequently repaired using an ipsilateral semitendinosus tendon. Two years later, the patient presented to our department with knee pain and loss of range of motion. Autogenous iliotibial band (ITB) enhancement combined with sagittal tendon lengthening plasty was used to improve the symptoms of the knee joint. The patient was followed up for 2 years. The knee joint function of the patient returned to the normal level. CONCLUSION: We successfully treated patella baja using autogenous ITB enhancement combined with sagittal tendon lengthening plasty.

Increased Expression of NXPH4 Correlates with Immune Cell Infiltration and Unfavorable Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading malignant carcinomas. Despite the advancement in the treatment for HCC, such as precise hepatectomy, radiotherapy, transarterial therapies, chemotherapy, targeted treatments, and immunotherapy, the 5-year overall survival rate of HCC is extremely low. Hence, novel biomarkers are urgently needed for advancing the therapy and prognosis of HCC. Neurexophilin 4 (NXPH4) is a neuropeptide-like glycoprotein. The study is designed to investigate the function of NXPH4 in HCC through a comprehensive bioinformatics analysis. NXPH4 expression status and prognostic values were analyzed via multiple datasets, such as TCGA, GEO, and ICGC. The association between NXPH4 and immune cell infiltration was estimated by TIMER, TISIDB, and CIBERSORT. In vitro, we explored the biological function of NXPH4 in JHH7 and SNU182 cells through knocking down the expression of NXPH4 via siRNA. In general, NXPH4 was predominantly upregulated in HCC tumors, and increased NXPH4 expression predicted unfavorable prognosis. The gene enrichment analysis displayed that NXPH4 was related with metabolic pathways. NXPH4 expression was correlated with immune cell infiltration. NXPH4 knockdown significantly suppressed proliferation, migration, and invasion of JHH7 and SNU182 cells. This study suggested that the upregulation of NXPH4 is associated with adverse prognosis and immune cell infiltration in HCC. NXPH4 could be a novel biomarker of unfavorable prognosis and an underlying target for immunotherapy in HCC.

Carboxylated fluorescent microsphere based immunochromatographic test strip enabled sensitive and quantitative on-site detection for florfenicol in eggs.

Florfenicol (FF), used popularly in prevention and treatment of virus infections in livestock and poultry, has widely been found in eggs and harmful to human health. In this work, a sensitive and quantitative on-site detecting solution, monoclonal antibody-based carboxylated fluorescent microsphere immunochromatographic test strip assay (FM-ICTS), is design and applied for FF detection. The proposed method can sensitively detect FF in low detection limit of 0.030 ng/g and quantitatively measure its concentration from 0.1 ng/mL to 8.1 ng/mL (R2 = 0.9991) with high repeatability (CV<8.0 %). In addition, the established FM-ICTS method exhibited high measurement accuracy in FF samples as compared with HPLC-MS analysis and demonstrated satisfied recoveries (99.1-101.3 %). More importantly, the quantitative FF test strip demonstrate ultra-high stability, which presents approximately equivalent detection ability to the fresh one after stored at 4 °C for more than one year or stored at 37 °C for 60 days. Therefore, the proposed method is a promising solution for rapidly and sensitively quantitative determination of FF in eggs.

Aluminium and nutrients induce changes in the profiles of phenolic substances in tea plants (Camellia sinensis CV TTES, No. 12 (TTE)).

BACKGROUND: Tea plants are always cultivated in acid soils in hilly regions and their growth can be dependent on to soluble aluminium (Al). The mechanism of Al detoxification and the influence of Al on phenolic compounds (i.e. catechin) in the roots of tea plants has remained obscure. This study aimed to investigate the influence of Al changes on the concentrations of phenolic substances in tea plants through hydroponic experiments. RESULTS: Tea plants were cultivated in nutrient solution containing 1.5 and 2.5 mmol L(-1) Al, and these treatments enhanced the growth of new buds and roots. Aluminium stimulated the uptake of Ca, Mg, K and Mn, whereas the uptake of Fe, Cu and Zn was retarded. Moreover, total phenol concentrations in tea plant tissues increased with increasing Al concentrations. In general, catechin concentrations in leaves increased with increasing Al concentrations in the hydroponic experiments. High correlation coefficients were obtained between Al and (-)-ECG (r(2) = 0.85, P < 0.01) and between Al and total phenols (r(2) = 0.92, P < 0.01). CONCLUSIONS: The Al concentration in tea plants indeed increases catechin concentrations and plays an important role in the growth of tea plants.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat.

BACKGROUND: There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model. RESULTS: Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. CONCLUSIONS: We found an increase in core-alpha-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.

-509C>T polymorphism in the TGF-beta1 gene promoter, impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-beta1 (TGF-beta1) is most frequently upregulated in tumor cells. The most studied -509C>T polymorphism of TGF-beta1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-beta1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. -509C>T polymorphism in the TGF-beta1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-beta1 mRNA expression levels. We also assessed the plasma TGF-beta1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position -509 of TGF-beta1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-beta1 or liver tumor tissue TGF-beta1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that -509C>T exchange in TGF-beta1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-beta1 or TGF-beta1 mRNA expression of liver tumor tissue.

Catechin transformation as influenced by aluminum.

Polyphenols (catechins) are vital biomolecules in tea plants (Camellia sinensis), which are well-known as typical Al accumulators. However, the interaction between Al and catechin remains obscured. The objective of the present study was to investigate the effect of Al on the transformation of (+)-catechin. Solutions with OH/Al molar ratios of 2.5 (pH 5.5) and 3.0 (pH 7.0) prepared at Al/catechin molar ratios (R) of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 were aged for 7 and 30 days, respectively. The precipitates were collected and examined by wet chemistry, X-ray diffraction, transmission electron microscopy, electron spin resonance (ESR), cross-polarization magic angle (CPMAS) 13C nuclear magnetic resonance (13C NMR) analyses, and Fourier transformation infrared absorption spectrometry (FT-IR). The weight of the precipitates increased with increasing Al/catechin molar ratios and with prolonged aging. The molar ratios of Al/catechin in the precipitates increased with increasing initial Al/catechin molar ratios and were close to the initial solution Al/catechin molar ratios. The chemical analysis and spectroscopic studies indicated that Al was bonded with catechin, forming a 1:1 type complex. The reaction of crystalline catechin with Al resulted in the formation of X-ray noncrystalline precipitates. The solid-state CPMAS 13C NMR spectra of the precipitates show the change in chemical shifts of catechin as a result of catechin complexation with Al. The FT-IR spectra of the Al-catechin precipitates also show the loss of absorption bands of several functional groups compared with catechin. The FT-IR data substantiate this reasoning. The ESR spectra of the precipitates show a single symmetrical line devoid of any fine splitting, indicating the presence of free radicals of semiquinones, which are commonly present in humified materials.

CYP1B1 and MYOC Mutations in Vietnamese Primary Congenital Glaucoma Patients.

PURPOSE: Primary congenital glaucoma (PCG, OMIM 231300), the most common glaucoma in infancy, is caused by developmental defects in the anterior chamber angle. The 3 implicated genes are cytochrome P450 family I subfamily B polypeptide 1 (CYP1B1), latent transforming growth factor ß-binding protein 2 (LTBP2), and myocilin (MYOC). In this study, we sought to determine CYP1B1 and MYOC sequence variations in a Vietnamese cohort of index cases with PCG and their families. METHODS: Thirty Vietnamese subjects with PCG and 120 normal Vietnamese subjects were recruited. PCG was defined by the presence of at least 2 of the following clinical manifestations: increased corneal diameter (>10 mm at birth), corneal edema, Haab's striae, optic disc changes, and absence of other ocular or systemic diseases associated with childhood glaucoma. The coding exons, intron and exon boundaries, and untranslated regions of CYP1B1 and MYOC genes were PCR amplified and subjected to bidirectional sequencing in all subjects. RESULTS: We identified 2 homozygous and 3 heterozygous CYP1B1 sequence alterations in our study subjects. Among the 5 mutations identified, 2 (p.H279L and p.L283F) were novel mutations, whereas 3 (p.A121_S122insDRPAFA, p.L107V, and p.V320L) had been previously reported in PCG cases. None of these mutations was observed in any of the 120 controls. Haplotypes generated with 6 non-disease-causing intragenic single nucleotide polymorphisms detected in CYP1B1 indicated that the most common haplotype in Vietnamese population is similar to that found in Chinese and Japanese. The genotype-phenotype correlation showed no significant difference between mutation and no-mutation groups for quantitative clinical features (presenting intraocular pressure, corneal diameter, number of surgeries performed, the cup-to-disc ratio) as well as for qualitative factors (bilateral cases, phenotype severity, and the prognosis) (P>0.05). CONCLUSIONS: Five out of 30 families with PCG (16.7%) had disease attributable to CYP1B1 alterations suggesting that CYP1B1 is not the major gene causing PCG in Vietnamese unlike in the case of Arab or Romany patients. This percentage is similar to that detected in studies of Japanese and Chinese patients with sporadic PCG. PCG has proven to be an ocular disease of genetic heterogeneity, calling for further studies to identify novel genes causing this disease.

Methylenetetrahydrofolate reductase gene polymorphism in endometrial cancer: A systematic review and meta-analysis.

OBJECTIVE: We conducted a meta-analysis of case-controlled prospective or retrospective studies to assess the effect of MTHFR polymorphisms on the risk of developing endometrial cancer. MATERIALS AND METHODS: PubMed, Cochrane, EMBASE, and ISI Web of Knowledge were searched (up to March 2014) for prospective or retrospective case-controlled studies that investigated the association of three MTHFR polymorphisms (rs180113 [C677T], rs1801131 [A1289C], and rs2274976 [G1793A]) with endometrial cancer. RESULTS: The patient population included subjects from three separate countries: China, Spain, and the USA. Only one study reported quantitative findings for MTHFR G1793A and, consequently, this polymorphism was not evaluated in our analysis. There were no significant associations of any MTHFR C677T or MTHFR A1298C alleles or genotypes with endometrial cancer (all p > 0.300). CONCLUSION: This meta-analysis does not support the association of endometrial cancer with two common MTHFR polymorphisms from this patient population.

[Aging affects early stage direction selectivity of MT cells in rhesus monkeys].

The middle temporal area (MT/V5) plays an important role in motion processing. Neurons in this area have a strongly selective response to the moving direction of objects and as such, the selectivity of MT neurons was proposed to be a neural mechanism for the perception of motion. Our previous studies have found degradation in direction selectivity of MT neurons in old monkeys, but this direction selectivity was calculated during the whole response time and the results were not able to uncover the mechanism of motion perception over a time course. Furthermore, experiments have found that direction selectivity was enhanced by attention at a later stage. Therefore, the response should be excluded in experiments with anesthesia. To further characterize the neural mechanism over a time course, we investigated the age-related changes of direction selectivity in the early stage by comparing the proportions of direction selective MT cells in old and young macaque monkeys using in vivo single-cell recording techniques. Our results show that the proportion of early-stage-direction-selective cells is lower in old monkeys than in young monkeys, and that the early stage direction bias (esDB) of old MT cells decreased relative to young MT cells. Furthermore, the proportion of MT cells having strong early stage direction selectivity in old monkeys was decreased. Accordingly, the functional degradation in the early stage of MT cells may mediate perceptual declines of old primates in visual motion tasks.

The effects of the renin-angiotensin-aldosterone system gene polymorphisms on insulin resistance in hypertensive families.

INTRODUCTION: The hereditability of insulin resistance has been demonstrated in both familial and twin studies. The effects of renin-angiotensin-aldosterone system gene polymorphisms on insulin resistance remain inconclusive. METHODS: This is a sibling-based association study. Polymorphisms of renin-angiotensin-aldosterone system genes were examined in 1113 hypertension and 676 normotension siblings from Chinese and Japanese hypertensive families. The generalized estimation equations method was used to compare the differences in metabolic variables between hypertension and normotensive siblings. RESULTS: For the G-6A polymorphism of AGT, GG siblings had lower 2-h insulin than siblings carrying the A allele (p=0.006). Siblings with different variants of the angiotensin II type 1 receptor A1166C had no difference in metabolic variables. Siblings carrying the D allele of the angiotensin converting enzyme gene had higher levels of fasting glucose, fasting insulin, area under the curve of insulin levels and the homeostasis model assessment of insulin resistance than II siblings (all p<0.05). Lower levels of fasting glucose and 2-h glucose were observed in siblings with the T allele than their CC homozygotes for the C-344T polymorphism of CYP11B2 (p<0.05). Siblings carrying three high-risk genotypes of the angiotensin converting enzyme, angiotensinogen and CYP11B2 had higher fasting glucose level than siblings carrying no high-risk genotypes (p=0.011). CONCLUSION: Our comprehensive analysis of renin-angiotensin-aldosterone system gene polymorphisms demonstrates that the angiotensin converting enzyme and CYP11B2 gene polymorphisms are associated with insulin resistance in hypertensive families.

No association of EGF 5'UTR variant A61G and hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection.

OBJECTIVE: Epidermal growth factor (EGF) has many biological functions, including mitogenesis, tumorigenesis, and proliferation of epidermal tissues. Previous studies have reported that the EGF +61 (A/G) single nucleotide polymorphism in the 5'-untranslated region of the EGF gene is functional, and is associated with development of hepatocellular carcinoma (HCC) in liver cirrhosis and various malignancy. Our aim was to investigate whether EGF gene A61G polymorphism could be implicated in susceptibility to and/or clinicopathological characteristics of HCC in Chinese patients with chronic hepatitis B virus (HBV) infection. METHODS: This polymorphism was studied in 387 patients with chronic HBV infection and in 208 healthy volunteers using restriction fragment-length polymorphism. The patients were divided into two groups: those without (n = 172) and those with HCC (n = 215). These 215 HCC patients with chronic HBV infection were designated as cases, and the remaining 172 patients without HCC served as controls. RESULTS: There were no significant differences in EGF genotype or allelic frequencies between cases and controls nor was EGF genotype or allelic frequencies associated with tumour number, size, growth phase, stage, and invasiveness. We also found ethnic heterogeneity in the functional EGF polymorphism. CONCLUSIONS: The present results show that although EGF gene A61G polymorphism is associated with development of HCC in liver cirrhosis, it is not sufficient for HCC in Chinese patients with chronic HBV infection.