Rituximab in combination with cyclosporine and steroid pulse therapy for childhood-onset multidrug-resistant nephrotic syndrome: a multicenter single-arm clinical trial (JSKDC11 trial).

BACKGROUND: Only 80% of children with idiopathic nephrotic syndrome respond well to glucocorticoid therapy. Multidrug-resistant nephrotic syndrome (MRNS) is associated with a poor kidney prognosis. Several retrospective studies have identified rituximab as an effective treatment for MRNS; however, prospective studies are required to assess its efficacy and safety. METHODS: We conducted a multicenter, non-blinded, single-arm trial to investigate the efficacy and safety of rituximab in patients with childhood-onset MRNS who were resistant to cyclosporine and more than three courses of steroid pulse therapy. The enrolled patients received four 375 mg/m2 doses of rituximab in combination with baseline cyclosporine and steroid pulse therapy. The primary endpoint was a > 50% reduction in the urinary protein/creatinine ratio from baseline on day 169. Complete and partial remissions were also evaluated. RESULTS: Six patients with childhood-onset MRNS were enrolled. All patients were negative for pathogenic variants of podocyte-related genes. On day 169, five patients (83.3%) showed a > 50% reduction in the urinary protein/creatinine ratio, two patients showed partial remission, and two patients showed complete remission. No deaths occurred and severe adverse events occurred in two patients (infection in one patient and acute kidney injury in one patient). Three patients needed treatment for moderate-to-severe infection. CONCLUSIONS: The study treatment effectively reduced the urinary protein/creatinine ratio in patients with childhood-onset MRNS. The adverse events in this study were within the expected range; however, attention should be paid to the occurrence of infections.

Development and validation of qPCR methods for nucleic acid biomarkers as a drug development tool: points to consider.

Nucleic acid (NA) biomarkers play critical roles in drug development. However, the global regulatory guidelines for assessing quantification methods specific to NA biomarkers are limited. The validation of analytical methods is crucial for the use of biomarkers in clinical and post-marketing evaluations of drug efficacy and adverse reactions. Given that quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) methods are the gold standards for the quantification of NA biomarkers, the Biomarker Analytical Method Validation Study Group in Japan has discussed considerations and made recommendations for the development and validation of qPCR- and RT-qPCR-based analytical methods for endogenous NA biomarkers as drug development tools. This white paper aims to contribute to the global harmonization of NA biomarker assay validation.

The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells.

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

Immunoglobulin G deposition on proximal tubules and the tubular basement membrane in acute tubular injury complicated with focal segmental glomerulosclerosis (FSGS): A possible prediction tool for subclinical FSGS.

Immunofluorescent deposition of immunoglobulin G (IgG) in the tubular basement membrane (TBM) has been evaluated in the diagnosis of various diseases; however, few studies have investigated the immunofluorescence of acute tubular injury (ATI). Herein, we attempted to clarify IgG expression in the proximal tubular epithelium and TBM in ATI due to various causes. Patients with ATI with nephrotic-range proteinuria, including focal segmental glomerulosclerosis (FSGS, n = 18) and minimal change nephrotic syndrome (MCNS, n = 8), ATI with ischemia (n = 6), and drug-induced ATI (n = 7), were enrolled. ATI was evaluated by light microscopy. CD15 and IgG double staining and IgG subclass staining were performed to evaluate immunoglobulin deposition in the proximal tubular epithelium and TBM. IgG deposition was identified in the proximal tubules only in the FSGS group. Furthermore, IgG deposition in the TBM was observed in the FSGS group showing severe ATI. IgG3 was predominantly deposited by the IgG subclass study. Our results indicate that IgG deposition in the proximal tubular epithelium and TBM suggests the leaking of IgG from the glomerular filtration barrier and its reabsorption by proximal tubules, which may predict disruption of the glomerular size barrier, including subclinical FSGS. FSGS with ATI should be included as a differential diagnosis when IgG deposition in TBM is observed.

Advanced Solid-State NMR Analysis of Two Crystal Forms of Ranitidine Hydrochloride: Detection of 1H-14N Intra-/Intermolecular Correlations.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The 1H-14N dipolar-based heteronuclear multiple quantum coherence (D-HMQC) analysis revealed the intermolecular correlation of Form 1 between the N atom of the nitro group and a proton of the furan moiety, which were closer than those of the intramolecular correlation reported using single X-ray crystal analysis. Thus, 1H-14N D-HMQC analysis could be useful for characterizing intermolecular interaction in ranitidine hydrochloride crystals. In addition, we reassigned the 13C solid-state NMR signals of ranitidine hydrochloride according to the liquid-state and multiple solid-state NMR experiments.

Systemic inflammation caused by an intracranial mesenchymal tumor with a EWSR1::CREM fusion presenting associated with IL-6/STAT3 signaling.

Pediatric neoplastic diseases account for about 10% of cases of fever of unknown origin (FUO), and most neoplastic disease cases are leukemia, lymphoma, and neuroblastoma. Brain tumors are rarely reported as the cause of FUO, although craniopharyngioma, metastatic brain tumor, and Castleman's disease have been reported. We report a case of intracranial mesenchymal tumor (IMT) with a FET:CREB fusion gene, which had inflammatory phenotype without neurological signs. A 10-year-old girl was admitted with a 2-month history of intermittent fever and headache, whereas her past history as well as her family history lacked special events. Sepsis work-up showed no pathological organism, and empirical antibiotic therapy was not effective. Bone marrow examination showed a negative result. Cerebrospinal fluid examination showed elevated protein as well as cell counts, and head magnaetic resonance imaging showed a hypervascular mass lesion with contrast enhancement in the left cerebellar hemisphere. The patient underwent tumor excision, which made the intermittent fever disappear. Pathological examinations resembled those of classic angiomatoid fibrous histiocytoma (AFH), but the morphological features were distinct from the AFH myxoid variant; then we performed break-apart fluorescence in situ hybridization and confirmed the tumor harbored the rare EWSR1::CREM fusion gene (Ewing sarcoma breakpoint region 1 gene (EWSR1) and cAMP response element binding (CREB) family gene). Consequently, we diagnosed the condition as IMT with EWSR1::CREM fusion. Elevated serum concentration of interleukin 6 (IL-6) was normalized after tumor resection, which suggested the fever could be caused by tumor-derived IL-6. This is the first case of IMT with EWSR1::CREM fusion that showed paraneoplastic symptoms associated with the IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Although brain tumors are rarely diagnosed as a responsible disease for FUO, they should be considered as a cause of unknown fever even in the absence of abnormal neurological findings.

Isolating Interaction-Null/Impaired Mutants using the Yeast Two-Hybrid Assay.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Flavonoids from Sedum japonicum subsp. oryzifolium (Crassulaceae).

Twenty-two flavonoids were isolated from the leaves and stems of Sedum japonicum subsp. oryzifolium (Crassulaceae). Of these compounds, five flavonoids were reported in nature for the first time, and identified as herbacetin 3-O-xyloside-8-O-glucoside, herbacetin 3-O-glucoside-8-O-(2'''-acetylxyloside), gossypetin 3-O-glucoside-8-O-arabinoside, gossypetin 3-O-glucoside-8-O-(2'''-acetylxyloside) and hibiscetin 3-O-glucoside-8-O-arabinoside via UV, HR-MS, LC-MS, acid hydrolysis and NMR. Other seventeen known flavonoids were identified as herbacetin 3-O-glucoside-8-O-arabinoside, herbacetin 3-O-glucoside-8-O-xyloside, gossypetin 3-O-glucoside-8-O-xyloside, quercetin, quercetin 3-O-glucoside, quercetin 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, kaempferol, kaempferol 3-O-glucoside, kaempferol 7-O-rhamnoside, kaempferol 3,7-di-O-rhamnoside, kaempferol 3-O-glucoside-7-O-rhamnoside, kaempferol 3-O-glucosyl-(1→2)-rhamnoside-7-O-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside, kaempferol 3-O-xylosyl-(1→2)-rhamnoside-7-O-rhamnoside, myricetin 3-O-glucoside and cyanidin 3-O-glucoside. Some flavonol 3,8-di-O-glycosides were found in Sedum japonicum subsp. oryzifolium as major flavonoids in this survey. They were presumed to be the diagnostic flavonoids in the species. Flavonoids were reported from S. japonicum for the first time.

Clinical, Pathological, and Genetic Characteristics in Patients with Focal Segmental Glomerulosclerosis.

Background: Approximately 30% of children with steroid-resistant nephrotic syndrome (SRNS) have causative monogenic variants. SRNS represents glomerular disease resulting from various etiologies, which lead to similar patterns of glomerular damage. Patients with SRNS mainly exhibit focal segmental glomerulosclerosis (FSGS). There is limited information regarding associations between histologic variants of FSGS (diagnosed using on the Columbia classification) and monogenic variant detection rates or clinical characteristics. Here, we report FSGS characteristics in a large population of affected patients. Methods: This retrospective study included 119 patients with FSGS, diagnosed using the Columbia classification; all had been referred to our hospital for genetic testing from 2016 to 2021. We conducted comprehensive gene screening of all patients using a targeted next-generation sequencing panel that included 62 podocyte-related genes. Data regarding patients' clinical characteristics and pathologic findings were obtained from referring clinicians. We analyzed the associations of histologic variants with clinical characteristics, kidney survival, and gene variant detection rates. Results: The distribution of histologic variants according to the Columbia classification was 45% (n=53) FSGS not otherwise specified, 21% (n=25) cellular, 15% (n=18) perihilar, 13% (n=16) collapsing, and 6% (n=7) tip. The median age at end stage kidney disease onset was 37 years; there were no differences in onset age among variants. We detected monogenic disease-causing variants involving 12 of the screened podocyte-related genes in 34% (40 of 119) of patients. The most common genes were WT1 (23%), INF2 (20%), TRPC6 (20%), and ACTN4 (10%). The perihilar and tip variants had the strongest and weakest associations with detection of monogenic variants (83% and 0%, respectively; P<0.001). Conclusions: We revealed the distributions of histologic variants of genetic FSGS and nongenetic FSGS in a large patient population. Detailed data concerning gene variants and pathologic findings are important for understanding the etiology of FSGS.

High Blood Pressure and Changes in the Body Mass Index Category Among Japanese Children: A Follow-Up Study Using the Updated American Academy of Pediatrics Guidelines.

Background High blood pressure (HBP) has become a public health issue worldwide. The relationship between high BP and changes in the body mass index (BMI) category in Japanese pubertal children has not yet been examined. To resolve this issue, we examined existing data with a focus on the primordial prevention of high BP signs, including elevated BP, among pubertal children aged 12 and 15 years. Methods Height, body weight, and BP data were examined from health checkups of 18,247 children conducted between 1993 and 2000 in the Karatsu Study, which was a cohort of pediatric lifestyle-related disease prevention medical health checkups in Japan. BP and BMI were assessed using the updated American Academy of Pediatrics (AAP) guidelines and Endocrine Society's clinical practice guidelines definitions, respectively. Results Follow-up data were obtained from 7,090 subjects (50.5% boys). Stage 2 hypertension (HTN) was detected in 3% and 2.7% of boys and girls aged 12 years, respectively, and in 2.7% and 1% of boys and girls aged 15 years, respectively. Among children aged 15 years, 1.4% were newly classified with stage 2 hypertension, and 15.6% exhibited improvements to a normal BP. A binomial logistic regression analysis of high BP and BMI category changes revealed odds ratios (OR) in the group with a deteriorated BMI category of 1.51 (95% confidence interval (CI), 1.17-1.94), 2.30 (95%CI, 1.66-3.17), and 6.83 (95%CI, 4.14-11.29) for elevated BP, stage 1 hypertension, and stage 2 hypertension, respectively. Conclusion High BP in puberty positively correlated with BMI category changes. Considering the presence of the tracking phenomenon in hypertension, BP monitoring is an essential part of the early strategy for the prevention of lifestyle-related diseases in childhood, and improvements in BP control are crucial in early life.

Dimerization of Firing Factors for Replication Origin Activation in Eukaryotes: A Crucial Process for Simultaneous Assembly of Bidirectional Replication Forks?

Controlling the activity of the heterohexameric Mcm2-7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2-7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a 'firing' reaction, in which the Mcm2-7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast Schizosaccharomyces pombe, Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.

Mycophenolate Mofetil after Rituximab for Childhood-Onset Complicated Frequently-Relapsing or Steroid-Dependent Nephrotic Syndrome.

BACKGROUND: Rituximab is the standard therapy for childhood-onset complicated frequently relapsing or steroid-dependent nephrotic syndrome (FRNS/SDNS). However, most patients redevelop FRNS/SDNS after peripheral B cell recovery. METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled trial to examine whether mycophenolate mofetil (MMF) administration after rituximab can prevent treatment failure (FRNS, SDNS, steroid resistance, or use of immunosuppressive agents or rituximab). In total, 39 patients (per group) were treated with rituximab, followed by either MMF or placebo until day 505 (treatment period). The primary outcome was time to treatment failure (TTF) throughout the treatment and follow-up periods (until day 505 for the last enrolled patient). RESULTS: TTFs were clinically but not statistically significantly longer among patients given MMF after rituximab than among patients receiving rituximab monotherapy (median, 784.0 versus 472.5 days, hazard ratio [HR], 0.59; 95% confidence interval [95% CI], 0.34 to 1.05, log-rank test: P=0.07). Because most patients in the MMF group presented with treatment failure after MMF discontinuation, we performed a post-hoc analysis limited to the treatment period and found that MMF after rituximab prolonged the TTF and decreased the risk of treatment failure by 80% (HR, 0.20; 95% CI, 0.08 to 0.50). Moreover, MMF after rituximab reduced the relapse rate and daily steroid dose during the treatment period by 74% and 57%, respectively. The frequency and severity of adverse events were similar in both groups. CONCLUSIONS: Administration of MMF after rituximab may sufficiently prevent the development of treatment failure and is well tolerated, although the relapse-preventing effect disappears after MMF discontinuation.

Analytical method validation for biomarkers as a drug development tool: points to consider.

Biomarkers are an important drug developmental tool. Assessment of quantitative analytical methods of biomarkers is not included in any regulatory documents in Japan. Use of biomarkers in clinical evaluations and supporting the post-marketing evaluation of drug efficacy and/or adverse reactions requires assessment and full validation of analytical methods for these biomarkers. The Biomarker Analytical Method Validation Study Group is a research group in Japan comprising industry and regulatory experts. Group members discussed and prepared this 'points to consider document' covering measurements of endogenous metabolites/peptides/proteins by ligand binding assays and chromatographic methods with or without mass spectrometry. We hope this document contributes to the global harmonization of biomarker assay validation.

A simple and rapid method to simultaneously analyze ciclesonide and its impurities in a ciclesonide metered-dose inhaler using on-line supercritical fluid extraction/supercritical fluid chromatography/quadrupole time-of-flight mass spectrometry.

A simple and rapid on-line SFE/SFC/quadrupole TOF-MS method to simultaneously analyze active pharmaceutical ingredients and impurities from metered-dose inhalers (MDIs) was developed using ciclesonide MDI (CIC-MDI) as an example. CIC-MDI, as drug Alvesco®, has been approved for the treatment of bronchial asthma, and its major impurities are listed in the European Pharmacopoeia and in the supplementary package inserts of Alvesco® (called as "Pharmaceutical interview form" in Japan). In the developed method, CIC-MDI was manually sprayed only once on a glass disc prior to the SFE/SFC/quadrupole TOF-MS. In the SFE, CIC and its impurities and other impurities having various polarities and hydrophobicity, were extracted in 3.5 min and subsequently separated on a CHIRALPAK IE-3 column to be detected by quadrupole TOF-MS in 6.5 min. This method would be applicable to the analysis of other inhalable pharmaceutical products whose sample preparation requires complicated procedures, as well as to the analysis of general pharmaceutical products for profiling impurities.

Quantitative Analysis of Ent-Kaurane Diterpenoids in Isodon Herb (Enmei-So) by HPLC-UV.

The terrestrial plants, Isodon japonicus (Burm. f.) H. Hara and Isodon trichocarpus (Maxim.) Kudô (Labiatae), are native to Japan. Different parts of these plants have been used as a traditional bitter stomachic, under the name Isodon herb (Enmei-so). Ent-kaurane diterpenoids are the major constituents of Isodon herb that contribute to the herb's medicinal properties. However, large variability with respect to the composition of these diterpenoids limits the suitability of Isodon herb as a pharmaceutical ingredient. Thus, an investigation of the factors that affect its chemical composition is required. In this study, the DNA-barcoding method, using internal transcribed spacer sequences of nuclear ribosomal DNA, was applied to cultivated and commercial samples of Isodon herb. Further, each such sample was separated into leaves, stems, and flowers and analyzed for diterpenoid content by HPLC. Moreover, the diterpenoid content in coarsely cut and powdered samples was evaluated. Results confirmed that the source species of these samples was I. japonicus or I. trichocarpus. The three major diterpenoids in Isodon herb were enmein, oridonin, and ponicidin. The diterpenoid content was affected by milling process. Moreover, the diterpenoid content was greatly affected by the ratio between leaves and stems in each sample. Thus, to accurately quantify the diterpenoids in Isodon herb, the use specific conditions such as drying using mild temperature conditions and avoiding milling of the samples might be necessary. This may help in regulating variations in the herb's composition, in turn, providing better quality and a safe herbal product for pharmaceutical use.

Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative.

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

Interaction of replication factor Sld3 and histone acetyl transferase Esa1 alleviates gene silencing and promotes the activation of late and dormant replication origins.

DNA replication in eukaryotes is a multi-step process that consists of three main reactions: helicase loading (licensing), helicase activation (firing), and nascent DNA synthesis (elongation). Although the contributions of some chromatin regulatory factors in the licensing and elongation reaction have been determined, their functions in the firing reaction remain elusive. In the budding yeast Saccharomyces cerevisiae, Sld3, Sld7, and Cdc45 (3-7-45) are rate-limiting in the firing reaction and simultaneous overexpression of 3-7-45 causes untimely activation of late and dormant replication origins. Here, we found that 3-7-45 overexpression not only activated dormant origins in the silenced locus, HMLα, but also exerted an anti-silencing effect at this locus. For these, interaction between Sld3 and Esa1, a conserved histone acetyltransferase, was responsible. Moreover, the Sld3-Esa1 interaction was required for the untimely activation of late origins. These results reveal the Sld3-Esa1 interaction as a novel level of regulation in the firing reaction.

Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae.

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.

DNA barcoding for identification of agarwood source species using trnL-trnF and matK DNA sequences.

Agarwood is a type of resinous wood found in the trunks of Aquilaria, Gonystylus, and Gyrinops species [1]. High-quality agarwood is extraordinarily expensive and therefore its source plant species have become depleted due to exploitation. In 2005, these species were added to Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [1]. Because these wild agarwood resources have become depleted, commercial production of agarwood has long been a desirable goal. In addition, inauthentic agarwood is sometimes produced from non-agarwood species. Few reports have attempted to identify source species in order to distinguish genuine from false agarwood. In this study, DNA was extracted from putative agarwood samples collected from Japanese, Indonesian, Thai, and Vietnamese markets. The trnL-trnF region and matK gene were amplified from each sample by PCR to serve as DNA barcodes for identifying the plant species to which each sample belonged. One of the wood samples did not originate from a genuine agarwood species. Although some species were identified, sequence data for agarwood source species currently available in GenBank is insufficient to identify the species to which all of these putative agarwood samples belonged. Thus, positive identification of remaining samples will require further exploration.