Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.

Actinobacterial chalkophores: the biosynthesis of hazimycins.

Copper is a transition metal element with significant effects on the morphological development and secondary metabolism of actinobacteria. In some microorganisms, copper-binding natural products are employed to modulate copper homeostasis, although their significance in actinobacteria remains largely unknown. Here, we identified the biosynthetic genes of the diisocyanide natural product hazimycin in Kitasatospora purpeofusca HV058, through gene knock-out and heterologous expression. Biochemical analyses revealed that hazimycin A specifically binds to copper, which diminishes its antimicrobial activity. The presence of a set of putative importer/exporter genes surrounding the biosynthetic genes suggested that hazimycin is a chalkophore that modulates the intracellular copper level. A bioinformatic survey of homologous gene cassettes, as well as the identification of two previously unknown hazimycin-producing Streptomyces strains, indicated that the isocyanide-based mechanism of copper homeostasis is prevalent in actinobacteria.

Endoscopic carbon dioxide insufflation tolerance test on the anal sphincter for anorectal hypofunction: a pilot and feasibility study.

Background: Anorectal function deteriorates with age. The diagnostic performance of the endoscopic pressure study integrated system (EPSIS), an endoscopic carbon dioxide (CO2) insufflation stress test of the lower esophageal sphincter has been previously evaluated as a diagnostic tool for gastroesophageal reflux disease. We aimed to evaluate the applicability of EPSIS in improving anorectal function. We hypothesized that EPSIS can be applied to the diagnosis of lower gastrointestinal tract disorders. Methods: This was a pilot, single-center, retrospective study using prospectively collected data between December 2021 and March 2022. It was designed to evaluate the differences in EPSIS rectal pressure measurements between older (≥80 years) and younger (<80 years) patients. At the end of the screening colonoscopy, the colonoscope was fixed in a retroflex position. When bowel movement was observed, CO2 was insufflated to the point where gas leakage occurred through the anus. The measured maximum pressure was defined as EPSIS-rectal pressure max (EPSIS-RP max) and compared between the groups. Results: Overall, 30 patients were included and examined. The median ages of the <80 and ≥80 years' groups were 53 (range: 27-79) and 82 (range: 80-94) years, respectively, with corresponding median measured EPSIS-RP max of 18.7 (range: 8.5-30.2) and 9.8 (range: 5.4-22.3) mmHg (P<0.001). Conclusions: Measurement of maximum rectal pressure illustrates the age-related decline in physiological anorectal function. Future studies should consider a loading test using EPSIS to quantify the decline in anorectal function and use it as a routine tool for screening and adjunctive diagnosis of anorectal hypofunction.

Clinical characteristics of absent contractility and ineffective esophageal motility: a multicenter study in Japan.

BACKGROUND AND AIM: Absent contractility (AC) and ineffective esophageal motility (IEM) are esophageal hypomotility disorders diagnosed using high-resolution manometry (HRM). Patient characteristics and disease course of these conditions and differential diagnosis between AC and achalasia are yet to be elucidated. METHODS: A multicenter study involving 10 high-volume hospitals was conducted. Starlet HRM findings were compared between AC and achalasia. Patient characteristics including underlying disorders and disease courses were analyzed in AC and IEM. RESULTS: Fifty-three patients with AC and 92 with IEM were diagnosed, while achalasia was diagnosed in 1784 patients using the Chicago classification v3.0 (CCv3.0). The cut-off integrated relaxation pressure (IRP) value at 15.7 mmHg showed maximum sensitivity (0.80) and specificity (0.87) for differential diagnosis of AC from type I achalasia. While most ACs were based on systemic disorders such as scleroderma (34%) and neuromuscular diseases (8%), 23% were sporadic cases. The symptom severity of AC was not higher than that of IEM. Regarding the diagnosis of IEM, the more stringent CCv4.0 excluded 14.1% of IEM patients than the CCv3.0, although patient characteristics did not change. In patients with the hypomotile esophagus, concomitance of reflux esophagitis was associated with low distal contractile integral and IRP values. AC and IEM transferred between each other, paralleling with the underlying disease course, although no transition to achalasia was observed. CONCLUSION: A successful determination of the optimal cut-off IRP value was achieved using the starlet HRM system to differentiate AC and achalasia. Follow-up HRM is also useful for differentiating AC from achalasia. Symptom severity may depend on underlying diseases instead of hypomotility severity.

Determination of esophageal squamous cell carcinoma and gastric adenocarcinoma on raw tissue using Raman spectroscopy.

BACKGROUND: Cancer detection is a global research focus, and novel, rapid, and label-free techniques are being developed for routine clinical practice. This has led to the development of new tools and techniques from the bench side to routine clinical practice. In this study, we present a method that uses Raman spectroscopy (RS) to detect cancer in unstained formalin-fixed, resected specimens of the esophagus and stomach. Our method can record a clear Raman-scattered light spectrum in these specimens, confirming that the Raman-scattered light spectrum changes because of the histological differences in the mucosal tissue. AIM: To evaluate the use of Raman-scattered light spectrum for detecting endoscop-ically resected specimens of esophageal squamous cell carcinoma (SCC) and gastric adenocarcinoma (AC). METHODS: We created a Raman device that is suitable for observing living tissues, and attempted to acquire Raman-scattered light spectra in endoscopically resected specimens of six esophageal tissues and 12 gastric tissues. We evaluated formalin-fixed tissues using this technique and captured shifts at multiple locations based on feasibility, ranging from six to 19 locations 200 microns apart in the vertical and horizontal directions. Furthermore, a correlation between the obtained Raman scattered light spectra and histopathological diagnosis was performed. RESULTS: We successfully obtained Raman scattered light spectra from all six esophageal and 12 gastric specimens. After data capture, the tissue specimens were sent for histopathological analysis for further processing because RS is a label-free methodology that does not cause tissue destruction or alterations. Based on data analysis of molecular-level substrates, we established cut-off values for the diagnosis of esophageal SCC and gastric AC. By analyzing specific Raman shifts, we developed an algorithm to identify the range of esophageal SCC and gastric AC with an accuracy close to that of histopathological diagnoses. CONCLUSION: Our technique provides qualitative information for real-time morphological diagnosis. However, further in vivo evaluations require an excitation light source with low human toxicity and large amounts of data for validation.

Learning curve for peroral endoscopic myotomy in therapeutic endoscopy experts and nonexperts: Large single-center experience.

OBJECTIVES: Reports on learning curve for peroral endoscopic myotomy (POEM) in therapeutic endoscopy nonexperts are limited. We aimed to assess the number of cases required to achieve POEM proficiency for endoscopic submucosal dissection (ESD) experts and nonexperts. METHODS: This is a retrospective study at the largest POEM referral center in Japan. POEM between April 2014 and December 2020 were included. Nonexperts and ESD experts were divided by training phases: A, 1-20; B, 21-40; C, 41-60; D, 61-80; and E, 81-100 cases. Primary outcome was operation time, and the phase to reach target time (83 min) was investigated. Secondary outcomes were clinical success rate, adverse events, and post-POEM gastroesophageal reflux disease (GERD). RESULTS: Five hundred and sixty-six cases were performed by 14 nonexperts, and 555 cases by 15 ESD experts. As the primary outcome, operation time in nonexperts was: A, 95 (79-115.8); B, 86.5 (71-105); C, 80 (70-100); D, 73 (64.5-100.5); and E, 73.5 (57.8-88.8) min, while in ESD experts: A, 90 (74-128); B, 77 (70-92); and C, 77 (70-93.5) min (median [interquartile range]). Operation time decreased significantly as experience increased in both groups (P < 0.001), and nonexperts required 41-60 cases to achieve proficiency, while experts required 21-40 cases. As secondary outcomes, in nonexperts, clinical success was 96.9-100%, adverse events were 5.0-9.2%, symptomatic GERD was 11.8-26.5%, and proton pump inhibitor (PPI) intake was 11.5-18.7% in each phase. While in experts, clinical success was 96.2-100%, adverse events were 3.0-5.8%, symptomatic GERD was 14.6-22.0%, and PPI intake was 12.6-17.9%. There were no significant differences among training phases. CONCLUSIONS: Non-ESD experts require more cases to achieve proficiency in POEM. These results are useful for establishing POEM training programs and institutional implementation of the procedure.

Association between endoscopic pressure study integrated system (EPSIS) and high-resolution manometry.

Background and study aims The endoscopic pressure study integrated system (EPSIS), a novel diagnostic tool for gastroesophageal reflux disease (GERD), allows evaluation of the anti-reflux barrier using endoscopy by monitoring the intragastric pressure (IGP) during insufflation. In this study, we evaluated the association between EPSIS results and lower esophageal sphincter (LES) function measured by high-resolution manometry (HRM) to elucidate whether EPSIS can evaluate the LES function. Patients and methods A retrospective, single-center study of patients with GERD symptoms who underwent endoscopy, pH-impedance monitoring, EPSIS, and HRM was conducted. The primary outcome was basal LES pressure and the secondary outcomes were end-respiratory LES pressure and integrated relaxation pressure (IRP). As EPSIS parameters, the following were measured: 1) pressure difference (mmHg), the difference between maximum and basal IGP; and 2) pressure gradient (mmHg/s), calculated by dividing pressure difference by the insufflating time. Pressure difference < 4.7 mmHg or pressure gradient < 0.07 mmHg/s was defined as an EPSIS GERD pattern. Results Forty-seven patients (median age: 53 years, 37 female) were analyzed. Pressure difference and pressure gradient significantly correlated with basal LES pressure (ρ = 0.29; P =  0.04 and ρ = 0.29; P =  0.04). Patients with EPSIS GERD pattern showed significantly lower basal LES pressure [13.2 (4.8-26.6) vs 25.3 (10.4-66.7) mmHg, P =  0.002], lower end-respiratory LES pressure [8.5 (1.1-15.9) vs 15.5 (1.9-43.9) mmHg, P =  0.019] and lower IRP [5.9 (1.0-12.0) vs 9.8 (1.3-17.8) mmHg, P =  0.020]. Conclusions This study showed a close association between EPSIS results and LES pressures measured by HRM. This indicates that EPSIS can evaluate the LES function during endoscopy and endorse the role of EPSIS as a diagnostic tool for GERD.

Discovery of prescopranone, a key intermediate in scopranone biosynthesis.

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Per-oral endoscopic myotomy as treatment for Killian-Jamieson diverticulum.

Killian-Jamieson diverticulum (KJD) is a rare type of esophageal diverticulum less commonly encountered compared with Zenker's diverticulum (ZD). Endoscopic approach for these diverticula has been rapidly evolving. Currently, a flexible endoscopic septum division is considered the first-line treatment for symptomatic ZD patients, however reported recurrence rates are over 10% according to recent literature. With the advent of submucosal tunneling technique established by per-oral endoscopic myotomy for achalasia, it has been applied to treat ZD named as Zenker's diverticulum per-oral endoscopic myotomy (Z-POEM) as a minimally invasive treatment. Although there are very few reports utilizing submucosal tunneling approach to KJD, we have opted to perform Z-POEM in order to safely perform complete dissection of the muscle septum while maintaining mucosal integration. Due to the difficulty of anatomical location of KJD, we created mucosal incision and subsequent submucosal tunnel directly at the level of the septum as opposed to creating a submucosal tunnel few centimeters proximal to the septum as being previously proposed. We report a case in which this technique was successfully performed with complete resolution of dysphagia without any adverse event. This technique permits to perform complete myotomies without the fear of causing perforation. Although larger cohorts are required to assess its safety and efficacy, Z-POEM to treat KJD seems to be promising.

Comparison of scope holding sign on endoscopy and lower esophageal sphincter contraction on high-resolution manometry: A pilot study.

Objectives: Lower esophageal sphincter (LES) plays a key role in gastroesophageal reflux disease (GERD) pathogenesis. In retroflexion and under sufficient insufflation, it can be seen how the lower esophagus grasps the endoscope, which we named scope holding sign (SHS). This study aimed to compare the SHS and LES pressure on high-resolution manometry (HRM), to elucidate whether the sphincter can be visualized endoscopically. Methods: This was a single-center, prospective pilot study. Patients with symptoms of GERD, who underwent endoscopy and HRM between February 2021 and April 2021, were included. A manometry catheter and an ultra-slim endoscope were inserted, and the resting LES pressure was measured. The lower esophagus holding (SHS-positive) and releasing (SHS-negative) the endoscope and catheter were observed. The LES pressures during SHS-positive and SHS-negative were compared. Results: Eleven patients (median age: 57 years; eight men) with normal esophageal motility were analyzed. The median LES pressure in SHS-positive was significantly higher than the resting LES pressure (40.4 [22.9-74.0] vs. 25.9 [2.0-66.7] mm Hg; p = 0.001) and the LES pressure in SHS-negative (4.6 [1.5-9.3]; p = 0.001). Furthermore, the LES pressure in SHS-negative was significantly lower than the resting LES pressure (4.6 [1.5-9.3] vs. 25.9 [2.0-66.7] mm Hg; p = 0.005). Conclusions: This study demonstrated that the SHS parallels LES pressure, indicating that the sphincter can be observed endoscopically. This may enable us to evaluate LES function during endoscopy in patients with GERD, thus, deserving further evaluation in future studies.

Treatment of achalasia with peroral endoscopic myotomy in situs inversus totalis.

Peroral endoscopic myotomy (POEM) has become established as a safe, effective, and versatile minimally invasive endoscopic treatment for achalasia and other esophageal motility disorders. Situs inversus totalis is a rare congenital disorder characterized by a completely reversed position (mirror-image) of the thoracic and abdominal visceral organs. This case report demonstrated a successful treatment of achalasia in a situs inversus totalis by POEM. Similar to the POEM procedure in a normal patient, it is important to maintain the orientation throughout the submucosal tunneling while keeping in mind the reversed orientation and anatomical landmarks. The submucosal tunnel and myotomy were created by an anterior approach which is in this case located at the reversed axis, at 10 o'clock position. There were no major technical modifications needed to be carried out by the operator. No adverse events were noted. Improvement in the Eckardt Symptom Score as well as the barium esophagogram and high-resolution manometry findings on 2-month follow-up exhibited that although POEM was performed in a reversed orientation, similar effects and outcomes were achieved, indicating a successful procedure in this case. In summary, by keeping in mind the reversed positioning and anatomical landmarks in situs inversus totalis, POEM shows to be a safe, effective, and versatile intervention in treating achalasia in situs inversus totalis without the need for major modifications in the procedural technique.

Achalasia with esophageal intramural hematoma treated by per-oral endoscopic myotomy (POEM).

Esophageal intramural hematoma (EIH) is a condition which occurs as a result of hemorrhage within the esophageal wall including the submucosal layer. However, reports of EIH on achalasia patients are quite limited and per-oral endoscopic myotomy (POEM) for achalasia with EIH has not been reported. This is the first case report that demonstrated a successful treatment of achalasia with EIH by POEM. In achalasia, since there is absence of lower esophageal sphincter relaxation, as barotraumatic pathogenesis, an increase in the intraesophageal pressure may cause EIH. As direct traumatic pathogenesis, the stasis of food may directly injure the esophageal wall resulting in EIH. After confirming the hematoma healed until it became an ulcer, POEM was performed on the posterior axis since the intramural hematoma was located anteriorly. The procedure was completed successfully without any occurrence of adverse events. On 2-months follow-up, improvement in dysphagia was noted, and complete epithelialization of the intramural hematoma region was seen on endoscopic examination. On 1-year follow-up, patient did not have recurrence of dysphagia and intramural hematoma. In summary, we reported a case of achalasia with EIH, which was treated by POEM. POEM procedure may be effective not only for the improvement of dysphagia but also for a better ulcer healing and prevention of intramural hematoma recurrence.

A novel endoscopic purse-string suture technique, "loop 9", for gastrointestinal defect closure: a pilot study.

BACKGROUND : This study aimed to assess the feasibility and efficacy of the novel loop 9 method of gastrointestinal (GI) defect closure. METHODS : 20 patients underwent a GI procedure that required defect closure. Loop 9 can be delivered through a single instrument channel (3.2 mm) and released at the defect site. After it has been anchored by two clips positioned on opposite sides of the defect edge, the loop 9 is tightened by pulling the end of the suture intraluminally using biopsy forceps. Additional clips are placed to achieve complete closure. The primary outcome was complete closure rate. The secondary outcomes were closure time, sustained closure rate, and adverse events. RESULTS : Complete closure was achieved in 100 % of cases. The mean size of the mucosal defects was 17.5 mm (range 10-55 mm). The median closure time was 14 minutes. The sustained closure rate was 90 %. No adverse events were noted. CONCLUSIONS : The loop 9 technique is feasible and effective in achieving complete and sustained closure of therapeutic endoscopy-related GI defects.

Novel modified side-by-side balloon dilatation method for esophagogastric junction strictures: Single-/double-barrel techniques.

Endoscopic balloon dilatation is an established treatment for benign gastrointestinal strictures. In order to dilatate safely, it is necessary to dilatate gradually and preferably under direct endoscopic visualization. However, it is currently difficult to achieve dilatation widths of 20-30 mm with commercial products and of over 30-mm with endoscopic visualization. Therefore, a safe and innovative application technique is needed for patients who need these 20-mm or greater dilatation widths. We have developed two methods that enable more than 20-mm dilatation under direct visualization. (i) Single-barrel method: an endoscopic balloon dilatation catheter was fixed with grasping forceps at the tip of the endoscope and carried into the stomach. The balloon catheter was released from the endoscope to create a retroflexed view. The endoscope and balloon were positioned adjacent to each other at the gastric cardia. The balloon was inflated gradually and the esophagogastric junction was dilatated under direct visualization. (ii) Double-barrel method: the same procedure was repeated with two balloons. Theoretically, we achieved a dilatation of >30-mm in diameter. These modified methods may be helpful for esophagogastric junction strictures, such as esophageal achalasia (serving as an initial endoscopic balloon dilatation prior to using a 30-mm achalasia balloon), or for functional strictures after Nissen fundoplication. In conclusion, our simple, modified method enables safe and gradual dilatation under direct endoscopic visualization. Additionally, combination of the sizes of one or two balloons can achieve various widths of dilatation.

Unified magnifying endoscopic classification for esophageal, gastric and colonic lesions: a feasibility pilot study.

Background and study aims Image-enhanced magnifying endoscopy allows optimization of the detection and diagnosis of lesions found in the gastrointestinal tract. Current organ-specific classifications are well-accepted by specialized endoscopists but may pose confusion for general gastroenterologists. To address this, our group proposed the Unified Magnifying Endoscopic Classification (UMEC) which can be applied either in esophagus, stomach, or colon. The aim of this study was to evaluate the diagnostic performance and clinical applicability of UMEC. Patients and methods A single-center, feasibility pilot study was conducted. Two endoscopists with experience in magnifying narrow band imaging (NBI), blinded to white-light and non-magnifying NBI findings as well as histopathological diagnosis, independently reviewed and diagnosed all images based on UMEC. In brief, UMEC is divided into three categories: non-neoplasia, intramucosal neoplasia, and deep submucosal invasive cancer. The diagnostic performance of UMEC was assessed while using the gold standard histopathology as a reference. Results A total of 303 gastrointestinal lesions (88 esophageal squamous lesions, 90 gastric lesions, 125 colonic lesions) were assessed. The overall accuracy for both endoscopists in the diagnosis of esophageal squamous cell cancer, gastric cancer, and colorectal cancer were 84.7 %, 89.5 %, and 83.2 %, respectively. The interobserver agreement for each organ, Kappa statistics of 0.51, 0.73, and 0.63, was good. Conclusions UMEC appears to be a simple and practically acceptable classification, particularly to general gastroenterologists, due to its good diagnostic accuracy, and deserves further evaluation in future studies.

Diagnosis of congenital esophageal stenosis in adults and treatment with peroral endoscopic myotomy.

BACKGROUND: Congenital esophageal stenosis (CES) in adults is a rare disorder that can present as achalasia, particularly in the distal esophagus. We describe the salient features of CES in adults and identify the feasibility and short-term outcomes of peroral endoscopic myotomy (POEM) for CES. METHODS: In this retrospective, single-center case series, we included 6 patients with a "misdiagnosis" of achalasia established elsewhere, ultimately diagnosed with CES and referred to our institution for POEM. Symptom improvement (clinical success rate), defined as an Eckardt Symptom Score (ESS) of <3 at 2-month follow up was assessed. RESULTS: Six patients (median age: 40 [range: 18-58] years; 4 males) were included. A long-standing history of dysphagia, ring-shaped stenosis on endoscopic examination, "lopsided hourglass" sign on barium esophagogram, and high-resolution manometry findings indicated by a compartmentalized intrabolus pressure pattern with distinction between the stenotic area and the lower esophageal sphincter were the salient features identified. POEM could not be completed in the first 2 cases due to technical challenges. All subsequent 4 patients who underwent successful POEM, exhibited improved ESS of ≤3 (clinical success rate 100%) at 2 months post-POEM. CONCLUSIONS: Along with identification of salient features on several diagnostic modalities, a differential diagnosis of CES in adults must be considered in patients presenting with long-standing history of dysphagia arising from childhood and persisting into adulthood. Although favorable short-term outcomes of POEM were achieved, further evaluation is still warranted, and an inexperienced operator should not attempt POEM on CES patients due to its technical difficulties.

Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression.

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.

Hemiacetal-less rapamycin derivatives designed and produced by genetic engineering of a type I polyketide synthase.

Engineering polyketide synthases is one of the most promising ways of producing a variety of polyketide derivatives. Exploring the undiscovered chemical space of this medicinally important class of middle molecular weight natural products will aid in the development of improved drugs in the future. In previous work, we established methodology designated 'module editing' to precisely manipulate polyketide synthase genes cloned in a bacterial artificial chromosome. Here, in the course of investigating the engineering capacity of the rapamycin PKS, novel rapamycin derivatives 1-4, which lack the hemiacetal moiety, were produced through the heterologous expression of engineered variants of the rapamycin PKS. Three kinds of module deletions in the polyketide synthase RapC were designed, and the genetically engineered vectors were prepared by the in vitro module editing technique. Streptomyces avermitilis SUKA34 transformed with these edited PKSs produced new rapamycin derivatives. The planar structures of 1-4 established based on 1D and 2D NMR, ESI-TOF-MS and UV spectra revealed that 2 and 3 had skeletons well-matched to the designs, but 1 and 4 did not. The observations provide important insights into the mechanisms of the later steps of rapamycin skeletal formation as well as the ketone-forming oxygenase RapJ.

Utility of a new automated diagnostic program in high-resolution esophageal manometry.

BACKGROUND: A new automated diagnostic program for high-resolution esophageal manometry (HREM) has been developed. This diagnostic program could detect locations of landmarks and could make final diagnoses automatically. However, the accuracy of the program is not known. The aim of this study was to evaluate the accuracy of the automated diagnostic program for HREM. METHODS: A total of 445 studies were enrolled. An HREM system (Starlet®) was used, and esophageal motility was diagnosed using the Chicago classification v3.0. First, the locations of the upper esophageal sphincter, transition zone, lower esophageal sphincter, esophago-gastric junction, crural diaphragm and stomach were determined, and each swallow was checked manually. Then, the parameters of the Chicago classification were calculated using an analytic program of the Starlet, and diagnoses were made by three experts. Second, all study raw data were analyzed again by the automated diagnostic program. Diagnoses made by the program were compared to those made by experts to evaluate the accuracy of the diagnoses. RESULTS: The new diagnostic program could identify the landmarks of each swallow, calculate the parameters and make a final diagnosis within 10 s. The diagnoses made by the automated diagnostic program were not matched to those made by experts in only 10 studies, and the overall accuracy of the new automated diagnostic program thus reached 97.8% (435/445). CONCLUSIONS: The new automated diagnostic program for HREM is clinically useful in terms of high diagnostic accuracy and time-saving.

Long-term clinical results of per-oral endoscopic myotomy (POEM) for achalasia: First report of more than 10-year patient experience as assessed with a questionnaire-based survey.

Background and study aims Since per-oral endoscopic myotomy (POEM) was introduced in 2010, it has become accepted as one of the standard treatments for esophageal achalasia worldwide. This study aimed to present long-term clinical results of POEM over 10 years and evaluate the technique and outcomes at the institution where it was first used in clinical settings. Patients and methods Questionnaire-based surveys were sent to patients who received POEM in our institution from September 2008 to May 2010. Patient demographics and procedural outcomes and open-ended questions were posed about the postoperative courses, including symptom improvement and recurrence, additional treatments, and post-POEM gastroesophageal reflux disease (GERD) symptoms. Achalasia symptoms and post-POEM GERD symptoms were evaluated with Eckhardt scores and GerdQ systems, respectively. Results Thirty-six consecutive POEMs were performed in that period and 10-year follow-up data were obtained from 15 patients (41.7 %). Although four cases (26.7 %) required additional pneumatic balloon dilatation (PBD), reduction in post-Eckardt scores were observed in 14 cases (93.3 %). GerdQ score was positive in one patient (6.7 %). Proton pump inhibitors (PPI) were taken by four patients (26.7 %) and their symptoms were well-controlled. Conclusions Clinical results of POEM over 10 years were favorable regardless of various factors. Symptoms improved even in patients who required additional treatments, suggesting that POEM plays a significant role in treatment of achalasia.