Hong-Hsing Liu

Background: Biliary atresia (BA) is the leading cause of pediatric liver transplantation. It is characterized by progressive extrahepatic bile duct obstruction in young infants. Inspired by the success of antifungal treatment in a newborn with BA-related obstructive cholangitis, we explored a potential link between BA and fungi, particularly Aspergillus . Fecal DNA was analyzed using 18S ribosomal sequencing and validated with a published fecal metagenomic dataset. Epidemiological data from the UK, Taiwan, and Japan were also examined. Results: Gut Aspergillus was exclusively detected in BA cases, suggesting it may be a potential trigger. Independent fecal metagenomic data from China and epidemiological correlations further supported this hypothesis. In the UK, BA presentations strongly correlated ( r = 0.98, 95% CI [0.36, 1.0], p = 0.02) with Aspergillosis , but not with Candidiasis , during the COVID-19 lockdown. In Taiwan, a decade of data showed BA incidence was significantly associated ( r = 0.78, 95% CI [0.29, 0.94], p = 0.01) with yearly Aspergillus -positive isolates among cancer-adjusted hospital admissions. In Japan, BA cases over 25 years correlated significantly ( r = 0.85, 95% CI [0.37, 0.97], p = 0.01) with visceral Aspergillus burdens in autopsied cases, but not with other fungal infections. Conclusions: The resolution of obstructive cholangitis in the antifungal-treated index case, together with multi-modal, cross-country evidence, highlights a potential link between gut Aspergillus and BA. Although limited by small sample size, retrospective design, and lack of mechanistic validation, the study may still be interpreted as hypothesis-generating and underscores the need for prospective studies to validate and extend these observations.

Up to two-thirds of biliary atresia (BA) patients need liver transplantation after the standard Kasai portoenterostomy treatment. Unrelenting cholangitis often precedes the full-blown presentation of BA. Using ribosomal 18S sequencing, here we report bowel colonization by non-commensal Aspergillus fungi in one case of neonatal obstructive cholangitis. Continuous oral fluconazole treatment resolved obstructive cholangitis completely. Colonization by non-commensal Aspergillus and Cerrena were additionally identified in 2 BA cases. In brief, bowel colonization by non-commensal fungi could be a treatable cause of neonatal obstructive cholangitis and potentially BA at an early stage.

DNA dumbbells have many applications. For example, they can be templates for rolling-circle amplification to produce highly accurate long-read sequences of single molecules. We have developed a highly efficient and simple method for ‘tunneling’ short-read Illumina sequencing libraries into long-read dumbbells. A 5’-exonuclease used in Gibson Assembly converted fixed Illumina P5/P7 sequences into sticky ends. Self-looping oligonucleotides with complementary 3’-overhangs were ligated to form dumbbells. The reaction proceeded in one pot and modified only end nucleotides of input libraries. Resulting dumbbells were ready for Phi29-dependent rolling circle amplification. Demonstrative readouts of full-length 16S rDNA from a standard microbial community, obtained on a Pacific Biosciences (PacBio) platform, confirmed successful tunneling of initially P5/P7-ended sequencing libraries. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on a PacBio platform.

Bowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10⁻⁴). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10⁻⁵) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients’ own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes Dengue fever an excellent disease model to explore. A principal component analyses (PCA)-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue dataset. Afterwards the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGFb1) and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in Dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of haemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schönlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGFb1-mediated class-switch would be a new factor to consider for infectious diseases.

The repertoire of IgG antibody responses to infection and vaccination varies depending on the characteristics of the immunogen and the ability of the host to mount a protective immune response. Chronic hepatitis B virus (HBV) infections are marked by persistent infection and immune tolerance to vaccination. This disease offers a unique opportunity to discover key repertoire signatures during infection and in response to vaccination. Complementarity determining region 3 of an antibody heavy chain (CDR-H3) has a major impact on the antigenic specificity of an antibody. We used next-generation sequencing to characterize the CDR-H3 sequences in paired siblings of 4 families in which only one member of each pair had chronic HBV infection. Blood samples were obtained before and 2 weeks after HBV vaccination. The analysis revealed a huge network of sequence-related CDR-H3 clones found almost exclusively among carriers. In contrast, vaccination induced significant increases of CDR-H3 cluster diversities among siblings without hepatitis B. Several vaccination-associated clone clusters were identified. Similar findings of vaccination-associated clone networks were observed in healthy adults receiving HBV boosters. These strategies can be used to identify signatures of other infectious diseases and accelerate discoveries of antibody sequences with important biomedical implications.