Cost-effectiveness analysis 3L of axicabtagene ciloleucel vs tisagenlecleucel and lisocabtagene maraleucel in Japan.

Aim: Cost-effectiveness analysis (CEA) was performed to compare axicabtagene ciloleucel (axi-cel) with tisagenlecleucel (tisa-cel) and lisocabtagene (liso-cel) for treatment of relapsed or refractory large B-cell lymphoma in adult patients after ≥2 lines of therapy in Japan. Materials & methods: Cost-effectiveness analysis was conducted using the partition survival mixture cure model based on the ZUMA-1 trial and adjusted to the JULIET and TRANSCEND trials using matching-adjusted indirect comparisons. Results & conclusion: Axi-cel was associated with greater incremental life years (3.13 and 2.85) and incremental quality-adjusted life-years (2.65 and 2.24), thus generated lower incremental direct medical costs (-$976.29 [-¥137,657] and -$242.00 [-¥34,122]), compared with tisa-cel and liso-cel. Axi-cel was cost-effective option compared with tisa-cel and liso-cel from a Japanese payer's perspective.

[Box: see text].

Assessment of metagenomic workflows using a newly constructed human gut microbiome mock community.

To quantify the biases introduced during human gut microbiome studies, analyzing an artificial mock community as the reference microbiome is indispensable. However, there are still limited resources for a mock community which well represents the human gut microbiome. Here, we constructed a novel mock community comprising the type strains of 18 major bacterial species in the human gut and assessed the influence of experimental and bioinformatics procedures on the 16S rRNA gene and shotgun metagenomic sequencing. We found that DNA extraction methods greatly affected the DNA yields and taxonomic composition of sequenced reads, and that some of the commonly used primers for 16S rRNA genes were prone to underestimate the abundance of some gut commensal taxa such as Erysipelotrichia, Verrucomicrobiota and Methanobacteriota. Binning of the assembled contigs of shotgun metagenomic sequences by MetaBAT2 produced phylogenetically consistent, less-contaminated bins with varied completeness. The ensemble approach of multiple binning tools by MetaWRAP can improve completeness but sometimes increases the contamination rate. Our benchmark study provides an important foundation for the interpretation of human gut microbiome data by providing means for standardization among gut microbiome data obtained with different methodologies and will facilitate further development of analytical methods.

Gut microbiota alternation under the intestinal epithelium-specific knockout of mouse Piga gene.

Crosstalk between the gut microbiota and intestinal epithelium shapes the gut environment and profoundly influences the intestinal immune homeostasis. Glycosylphosphatidylinositol anchored proteins (GPI - APs) contribute to a variety of gut-associated immune functions, including microbial surveillance and defense, and epithelial cell polarity. Properly polarised epithelial cells are essential for the establishment of the barrier function of gut epithelia. The Piga gene is one among seven genes that encode for an enzyme which is involved in the first step of GPI-anchor biosynthesis. This is the first study reporting a knockout of the intestinal epithelial cell-specific Piga gene (Piga-/-) and its association with the gut microbiota in mice using a whole metagenome shotgun-based sequencing approach. An overall reduced microbiota diversity has been observed in the Piga-/- group as compared to the control group (ANOVA p = 0.34). The taxonomic biomarkers, namely: Gammaproteobacteria (class), Enterobacterales (order), Enterobacteriaceae (family), Escherichia (genus), Proteus (genus) and Escherichia coli (species), increased more in the Piga-/- mice as compared to in the control group. Further, the pathogenic E. coli strains, namely E. coli O157:H7 str. EDL 933 (EHEC), E. coli CFT073 (UPEC) and E. coli 536 (UPEC), were found in the Piga-/- mice which also harbored virulence factor transporters. In addition, the taxa responsible for short chain fatty acid production were decreased in the Piga-/- group. The Piga-/- mice gut harbored an increased number of microbial functions responsible for the survival of pathogens in the inflamed gut environment. Our observations clearly indicate that the Piga-/- mice gut might have an overall enhancement in pathogenic behaviour and reduced capabilities beneficial to health.

Shotgun metagenomic sequencing revealed the prebiotic potential of a grain-based diet in mice.

In the present study, we elucidated the effect of grain-based (GB) diet containing both soluble and insoluble fibers and purified ingredients-based (PIB) diet containing only insoluble fiber, namely cellulose on mice gut microbiome using whole shotgun based metagenomic sequencing. Although the fiber content in both diet types is the same (5%) the presence of soluble fiber only in the GB diet differentiates it from the PIB diet. The taxonomic analysis of sequenced reads reveals a significantly higher enrichment of probiotic Lactobacilli in the GB group as compared to the PIB group. Further, the enhancement of energy expensive cellular processes namely, cell cycle control, cell division, chromosome partitioning, and transcription is observed in the GB group which could be due to the metabolization of the soluble fiber for faster energy production. In contrast, a higher abundance of cellulolytic bacterial community namely, the members of family Lachnospiraceae and Ruminococcaceae and the metabolism functions are found in the PIB group. The PIB group shows a significant increase in host-derived oligosaccharide metabolism functions indicating that they might first target the host-derived oligosaccharides and self-stored glycogen in addition to utilising the available cellulose. In addition to the beneficial microbial community variations, both the groups also exhibited an increased abundance of opportunistic pathobionts which could be due to an overall low amount of fiber in the diet. Furthermore, backtracing analysis identified probiotic members of Lactobacillus, viz., L. crispatus ST1, L. fermentum CECT 5716, L. gasseri ATCC 33323, L. johnsonii NCC 533 and L. reuteri 100-23 in the GB group, while Bilophila wadsworthia 3_1_6, Desulfovibrio piger ATCC 29098, Clostridium symbiosum WAL-14163, and Ruminococcaceae bacterium D16 in the PIB group. These data suggest that Lactobacilli, a probiotic community of microorganisms, are the predominant functional contributors in the gut of GB diet-fed mice, whereas pathobionts too coexisted with commensals in the gut microbiome of the PIB group. Thus at 5% fiber, GB modifies the gut microbial ecology more effectively than PIB and the inclusion of soluble fiber in the GB diet may be one of the primary factors responsible for this impact.

Impact of dietary fructooligosaccharides (FOS) on murine gut microbiota and intestinal IgA secretion.

Fructooligosaccharides (FOS) are considered as prebiotics and are well known for their health-promoting properties, including antitumor, allergy-preventive, and infection-protective effects. They exert these effects by modulating the gut microbial composition and dynamics. In the present study, we performed a comparative whole metagenome shotgun sequencing analysis (WMGS) to elucidate the gut microbiota and secretary Immunoglobulin A (SIgA) dynamics as a result of 5% (w/w) FOS supplementation over a period of 7 days (fecal samples were collected every day). A number of taxa including Bacteroides, Lactobacillus, Roseburia, Clostridia, Faecalibaculum, and Enterorhabdus were found to be modulated with SIgA production in the murine gut. The process of SIgA production from FOS metabolization was found to be carried out via the production of short-chain fatty acids in the gut. Species of Bacteroides and Roseburia; namely, B. caccae, B. finegoldii, B. ovatus, B. thetaiotamicron, and Roseburia intestinalis, respectively, are predominantly responsible for FOS metabolization in the murine gut. The abundances of these bacterial species and their corresponding functions involved in FOS metabolization decreased over time even though these prebiotics were administered continuously for seven days. This suggests that there is a decrease in FOS metabolization over time. In addition, the present analysis suggests that the administration of FOS may help to reduce the pathogenic bacteria from the gut via SIgA production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03116-3.

Gut microorganisms act together to exacerbate inflammation in spinal cords.

Accumulating evidence indicates that gut microorganisms have a pathogenic role in autoimmune diseases, including in multiple sclerosis1. Studies of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis)2,3, as well as human studies4-6, have implicated gut microorganisms in the development or severity of multiple sclerosis. However, it remains unclear how gut microorganisms act on the inflammation of extra-intestinal tissues such as the spinal cord. Here we show that two distinct signals from gut microorganisms coordinately activate autoreactive T cells in the small intestine that respond specifically to myelin oligodendrocyte glycoprotein (MOG). After induction of experimental autoimmune encephalomyelitis in mice, MOG-specific CD4+ T cells are observed in the small intestine. Experiments using germ-free mice that were monocolonized with microorganisms from the small intestine demonstrated that a newly isolated strain in the family Erysipelotrichaceae acts similarly to an adjuvant to enhance the responses of T helper 17 cells. Shotgun sequencing of the contents of the small intestine revealed a strain of Lactobacillus reuteri that possesses peptides that potentially mimic MOG. Mice that were co-colonized with these two strains showed experimental autoimmune encephalomyelitis symptoms that were more severe than those of germ-free or monocolonized mice. These data suggest that the synergistic effects that result from the presence of these microorganisms should be considered in the pathogenicity of multiple sclerosis, and that further study of these microorganisms may lead to preventive strategies for this disease.

Association of colitis with gut-microbiota dysbiosis in clathrin adapter AP-1B knockout mice.

Inflammatory bowel disease results from alterations in the immune system and intestinal microbiota. The role of intestinal epithelial cells (IECs) in maintaining gut homeostasis is well known and its perturbation often causes gastrointestinal disorders including IBD. The epithelial specific adaptor protein (AP)-1B is involved in the establishment of the polarity of IECs. Deficiency of the AP-1B µ subunit (Ap1m2-/-) leads to the development of chronic colitis in mice. However, how this deficiency affects the gut microbes and its potential functions remains elusive. To gain insights into the gut microbiome of Ap1m2-/- mice having the colitis phenotype, we undertook shotgun metagenomic sequencing analysis of knockout mice. We found important links to the microbial features involved in altering various physiological pathways, including carbohydrate metabolism, nutrient transportation, oxidative stress, and bacterial pathogenesis (cell motility). In addition, an increased abundance of sulfur-reducing and lactate-producing bacteria has been observed which may aggravate the colitis condition.

Lactobacillus plantarum USM8613 Aids in Wound Healing and Suppresses Staphylococcus aureus Infection at Wound Sites.

This study aimed to elucidate the targets and mechanisms of anti-staphylococcal effects from bioactive metabolites produced by lactic acid bacteria. We aimed to better understand the safety and efficacy of these bioactive metabolites in in vivo systems, typically at topical sites. The cell-free supernatant and protein-rich fraction from Lactobacillus plantarum USM8613 inhibited staphyloxanthin biosynthesis, reduced (p < 0.05) the cell number of Staphylococcus aureus by 106 CFU/mL and reduced biofilm thickness by 55% in S. aureus-infected porcine skins. Genome-wide analysis and gene expression analysis illustrated the production of several plantaricins, especially the plantaricins EF and JK that enhanced the anti-staphylococcal effects of L. plantarum USM8613. In vivo data using rats showed that the protein-rich fraction from L. plantarum USM8613 exerted wound healing properties via direct inhibition of S. aureus and promoted innate immunity, in which the expression of ß-defensin was significantly (p < 0.05) upregulated by 3.8-fold. The protein fraction from L. plantarum USM8613 also significantly enhanced (p < 0.05) the production of cytokines and chemokines through various stages of wound recovery. Using ∆atl S. aureus, the protein-rich fraction from L. plantarum USM8613 exerted inhibitory activity via targeting the atl gene in S. aureus. Taken altogether, our present study illustrates the potential of L. plantarum USM8613 in aiding wound healing, suppressing of S. aureus infection at wound sites and promoting host innate immunity.

Extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase attributed to the anti-staphylococcal activity of Lactobacillus plantarum USM8613.

Increasing levels of antibiotic resistance in pathogens, including Staphylococcus aureus, remains a serious problem for public health, leading to the need for better alternative antimicrobial strategies. The antimicrobial proteins produced by Lactobacillus plantarum USM8613 attributed to its anti-staphylococcal activity were identified as extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase (GADPH), both with different mechanisms of action. Extracellular transglycosylase, which contains a LysM domain, exerts a cell wall-mediated killing mechanism, while GADPH penetrates into S. aureus cells and subsequently induces the overexpression of autolysis regulators, resulting in S. aureus autolysis. Both extracellular transglycosylase and GADPH exert anti-inflammatory effects in S. aureus-infected HaCaT cells by reducing the expression and production of TLR-2, hBDs and various pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α, and IL-8). Taken together, extracellular transglycosylase and GADPH produced by L. plantarum USM8613 could potentially be applied as an alternative therapeutic agent to treat S. aureus skin infections and promote skin health.

An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm.

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.

Lactobacillus fermentum FTDC 8312 combats hypercholesterolemia via alteration of gut microbiota.

In this study, hypercholesterolemic mice fed with Lactobacillus fermentum FTDC 8312 after a seven-week feeding trial showed a reduction in serum total cholesterol (TC) levels, accompanied by a decrease in serum low-density lipoprotein cholesterol (LDL-C) levels, an increase in serum high-density lipoprotein cholesterol (HDL-C) levels, and a decreased ratio of apoB100:apoA1 when compared to those fed with control or a type strain, L. fermentum JCM 1173. These have contributed to a decrease in atherogenic indices (TC/HDL-C) of mice on the FTDC 8312 diet. Serum triglyceride (TG) levels of mice fed with FTDC 8312 and JCM 1173 were comparable to those of the controls. A decreased ratio of cholesterol and phospholipids (C/P) was also observed for mice fed with FTDC 8312, leading to a decreased number of spur red blood cells (RBC) formation in mice. Additionally, there was an increase in fecal TC, TG, and total bile acid levels in mice on FTDC 8312 diet compared to those with JCM 1173 and controls. The administration of FTDC 8312 also altered the gut microbiota population such as an increase in the members of genera Akkermansia and Oscillospira, affecting lipid metabolism and fecal bile excretion in the mice. Overall, we demonstrated that FTDC 8312 exerted a cholesterol lowering effect that may be attributed to gut microbiota modulation.

Whole Exome Sequencing in Eight Thai Patients With Leber Congenital Amaurosis Reveals Mutations in the CTNNA1 and CYP4V2 Genes.

Purpose: Our goal was to describe the clinical and molecular genetic findings in Thai patients with Leber congenital amaurosis (LCA). Methods: Whole exome sequencing (WES) was performed in eight unrelated patients. All genes responsible for inherited retinal diseases (IRDs) based on RetNet were selected for analysis. Potentially causative variants were filtered through a bioinformatics pipeline and validated using Sanger sequencing. Segregation analysis of the causative genes was performed in family members when available. Results: Eleven deleterious variants, six nonsense and five missense, were identified in seven genes: four LCA-associated genes (CEP290, IQCB1, NMNAT1, and RPGRIP1), one gene responsible for syndromic LCA (ALMS1), and two IRDs-related genes (CTNNA1 and CYP4V2). Clinical reassessment supported the diagnosis of syndromic LCA in those patients harboring potentially pathogenic variants in the ALMS1. Interestingly, two causative genes, CTNNA1 and CYP4V2, previously reported to cause butterfly-shaped pigment dystrophy (BSPD) and Bietti's crystalline dystrophy (BCD), respectively, were detected in two other patients. These two patients developed rapid and severe visual loss in contrast to BSPD and BCD patients in previous studies. The results of this study demonstrate that causative variants identified in the CTNNA1 and CYP4V2 genes are also associated with LCA. Conclusions: This is the first report describing the molecular genetics and clinical manifestations of Thai patients with LCA. The present study expands the spectrum of LCA-associated genes, which is a benefit for molecular diagnosis. The identification of mutations in the CTNNA1 and CYP4V2 genes requires further elucidation in larger cohorts with LCA.

Comparative metagenomic analysis of PAH degradation in soil by a mixed microbial consortium.

In this study, we used a taxonomic and functional metagenomic approach to analyze some of the effects (e.g. displacement, permanence, disappearance) produced between native microbiota and a previously constructed Polycyclic Aromatic Hydrocarbon (PAH)-degrading microbial consortium during the bioremediation process of a soil polluted with PAHs. Bioaugmentation with a fungal-bacterial consortium and biostimulation of native microbiota using corn stover as texturizer produced appreciable changes in the microbial diversity of polluted soils, shifting native microbial communities in favor of degrading specific populations. Functional metagenomics showed changes in gene abundance suggesting a bias towards aromatic hydrocarbon and intermediary degradation pathways, which greatly favored PAH mineralization. In contrast, pathways favoring the formation of toxic intermediates such as cytochrome P450-mediated reactions were found to be significantly reduced in bioaugmented soils. PAH biodegradation in soil using the microbial consortium was faster and reached higher degradation values (84% after 30 d) as a result of an increased co-metabolic degradation when compared with other mixed microbial consortia. The main differences between inoculated and non-inoculated soils were observed in aromatic ring-hydroxylating dioxygenases, laccase, protocatechuate, salicylate and benzoate-degrading enzyme genes. Based on our results, we propose that several concurrent metabolic pathways are taking place in soils during PAH degradation.

MetaTreeMap: An Alternative Visualization Method for Displaying Metagenomic Phylogenic Trees.

Metagenomic samples can contain hundreds or thousands of different species. The most common method to identify these species is to sequence the samples and then classify the reads to nodes along a phylogenic tree. Linear representations of trees with so many nodes face legibility issues. In addition, such views are not optimal for appreciating the read quantity assigned to each node. The problem is exaggerated when comparison between multiple samples is needed. MetaTreeMap adapts a visualization method that addresses these weaknesses. The tree is represented by nested rectangles that illustrate the number or percentage of assigned reads. MetaTreeMap implements various options specific to phylogenic trees that allow for quick overview and investigation of the information. More generally, the goal of this software is to provide the user with the ability to easily display phylogenic trees based on various quantities assigned to the nodes, such as read number, percentage or other values. The tool can be used online at http://metasystems.riken.jp/visualization/treemap/.

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis.

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis's capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations characterized by progressive loss of photoreceptor cells and RPE functions. More than 70 causative genes are known to be responsible for RP. This study aimed to identify the causative gene in a patient from a consanguineous family with childhood-onset severe retinal dystrophy. METHODS: To identify the defective gene, whole exome sequencing was performed. Candidate causative variants were selected and validated using Sanger sequencing. Segregation analysis of the causative gene was performed in additional family members. To verify that the mutation has an effect on protein synthesis, an expression vector containing the first ten amino acids of the mutant protein fused with the DsRed2 fluorescent protein was constructed and transfected into HEK293T cells. Expression of the fusion protein in the transfected cells was measured using fluorescence microscopy. RESULTS: By filtering against public variant databases, a novel homozygous missense mutation (c.3G>A) localized in the start codon of the MERTK gene was detected as a potentially pathogenic mutation for autosomal recessive RP. The c.3G>A mutation cosegregated with the disease phenotype in the family. No expression of the first ten amino acids of the MerTK mutant fused with the DsRed2 fluorescent protein was detected in HEK293T cells, indicating that the mutation affects the translation initiation site of the gene that may lead to loss of function of the MerTK signaling pathway. CONCLUSIONS: We report a novel missense mutation (c.3G>A, p.0?) in the MERTK gene that causes severe vision impairment in a patient. Taken together with previous reports, our results expand the spectrum of MERTK mutations and extend our understanding of the role of the MerTK protein in the pathogenesis of retinitis pigmentosa.

Comparative genome analyses of novel Mangrovimonas-like strains isolated from estuarine mangrove sediments reveal xylan and arabinan utilization genes.

To date, the genus Mangrovimonas consists of only one species, Mangrovimonas yunxiaonensis strain LY01 that is known to have algicidal effects against harmful algal blooms (HABs) of Alexandrium tamarense. In this study, the whole genome sequence of three Mangrovimonas-like strains, TPBH4(T)(=LMG 28913(T),=JCM 30882(T)), ST2L12(T)(=LMG 28914(T),=JCM 30880(T)) and ST2L15(T)(=LMG 28915(T),=JCM 30881(T)) isolated from estuarine mangrove sediments in Perak, Malaysia were described. The sequenced genomes had a range of assembly size ranging from 3.56 Mb to 4.15 Mb which are significantly larger than that of M. yunxiaonensis LY01 (2.67 Mb). Xylan, xylose, L-arabinan and L-arabinose utilization genes were found in the genome sequences of the three Mangrovimonas-like strains described in this study. In contrast, these carbohydrate metabolism genes were not found in the genome sequence of LY01. In addition, TPBH4(T) and ST2L12(T) show capability to degrade xylan using qualitative plate assay method.

Complete genome sequence of Streptomyces sp. strain CFMR 7, a natural rubber degrading actinomycete isolated from Penang, Malaysia.

Streptomyces sp. strain CFMR 7, which naturally degrades rubber, was isolated from a rubber plantation. Whole genome sequencing and assembly resulted in 2 contigs with total genome size of 8.248 Mb. Two latex clearing protein (lcp) genes which are responsible for rubber degrading activities were identified.

Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater.

BACKGROUND: Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. RESULTS: A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). CONCLUSIONS: A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.

Whole exome sequencing in Thai patients with retinitis pigmentosa reveals novel mutations in six genes.

PURPOSE: To identify disease-causing mutations and describe genotype-phenotype correlations in Thai patients with nonsyndromic retinitis pigmentosa (RP). METHODS: Whole exome sequencing was performed in 20 unrelated patients. Eighty-six genes associated with RP, Leber congenital amaurosis, and cone-rod dystrophy were analyzed for variant detection. RESULTS: Seventeen variants (13 novel and 4 known) in 13 genes were identified in 11 patients. These variants include 10 missense substitutions, 2 nonsense mutations, 3 deletions, 1 insertion, and 1 splice site change. Nine patients with identified inheritance patterns carried a total of 10 potentially pathogenic mutations located in genes CRB1, C8orf37, EYS, PROM1, RP2, and USH2A. Three of the nine patients also demonstrated additional heterozygous variants in genes ABCA4, GUCY2D, RD3, ROM1, and TULP1. In addition, two patients carried variants of uncertain significance in genes FSCN2 and NR2E3. The RP phenotypes of our patients were consistent with previous reports. CONCLUSIONS: This is the first report of mutations in Thai RP patients. These findings are useful for genotype-phenotype comparisons among different ethnic groups.