Long-read sequencing revealing intragenic deletions in exome-negative spastic paraplegias.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and weakness in the lower extremities. To date, a total of 88 types of SPG are known. To diagnose HSP, multiple technologies, including microarray, direct sequencing, multiplex ligation-dependent probe amplification, and short-read next-generation sequencing, are often chosen based on the frequency of HSP subtypes. Exome sequencing (ES) is commonly used. We used ES to analyze ten cases of HSP from eight families. We identified pathogenic variants in three cases (from three different families); however, we were unable to determine the cause of the other seven cases using ES. We therefore applied long-read sequencing to the seven undetermined HSP cases (from five families). We detected intragenic deletions within the SPAST gene in four families, and a deletion within PSEN1 in the remaining family. The size of the deletion ranged from 4.7 to 12.5 kb and involved 1-7 exons. All deletions were entirely included in one long read. We retrospectively performed an ES-based copy number variation analysis focusing on pathogenic deletions, but were not able to accurately detect these deletions. This study demonstrated the efficiency of long-read sequencing in detecting intragenic pathogenic deletions in ES-negative HSP patients.

Repeat conformation heterogeneity in cerebellar ataxia, neuropathy, vestibular areflexia syndrome.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.

Acute Adverse Events at a Mass Vaccination Site after the Third and Fourth COVID-19 Vaccinations in Japan.

The third and fourth doses of the vaccine against coronavirus disease 2019 (COVID-19) were widely administered in Japan since December 2021. Currently, however, data are scarce regarding acute adverse events with the third and fourth doses. The present study reports the profiles of acute adverse events after the third and fourth COVID-19 vaccine doses, seen at the site of a mass vaccination center in Japan. Between December 2021 and July 2022, 267,515 individuals received the third, and 32,934 received the fourth COVID-19 vaccine dose at the mass vaccination center, of whom 442 recipients of the third (0.19%), and 22 recipients of the fourth (0.07%) dose reported acute adverse events and were examined by doctors on site. The most common diagnosis was vasovagal syncope/presyncope (incidence: 0.01-0.10%), followed by other miscellaneous complaints, acute allergic reactions (0.05-0.005%), and anaphylaxis (< 0.005%). Vasovagal syncope/presyncope occurred most frequently in recipients in those in their 20s, whereas acute allergic reactions were most frequent in those in their 40s. Both reactions were more frequent in women than men. The peak occurrence of vasovagal syncope/presyncope was earlier than 15 min after the injection, whereas that of acute allergic reaction was later than 15 min after the injection. The incidence of acute allergic reactions appeared to differ between various vaccine manufacturers, whereas that of vasovagal syncope/presyncope did not. These real-world data may benefit the safe and efficient implementation of mass vaccination campaigns for citizens who want to receive COVID-19 vaccines now and in the future.

Warm Temperature Promotes Shoot Regeneration in Arabidopsis thaliana.

Many plants are able to regenerate upon cutting, and this process can be enhanced in vitro by incubating explants on hormone-supplemented media. While such protocols have been used for decades, little is known about the molecular details of how incubation conditions influence their efficiency. In this study, we find that warm temperature promotes both callus formation and shoot regeneration in Arabidopsis thaliana. We show that such an increase in shoot regenerative capacity at higher temperatures correlates with the enhanced expression of several regeneration-associated genes, such as CUP-SHAPED COTYLEDON 1 (CUC1) encoding a transcription factor involved in shoot meristem formation and YUCCAs (YUCs) encoding auxin biosynthesis enzymes. ChIP-sequencing analyses further reveal that histone variant H2A.Z is enriched on these loci at 17°C, while its occupancy is reduced by an increase in ambient temperature to 27°C. Moreover, we provide genetic evidence to demonstrate that H2A.Z acts as a repressor of de novo shoot organogenesis since H2A.Z-depleted mutants display enhanced shoot regeneration. This study thus uncovers a new chromatin-based mechanism that influences hormone-induced regeneration and additionally highlights incubation temperature as a key parameter for optimizing in vitro tissue culture.

Bone-patellar tendon-bone autograft maturation is superior to double-bundle hamstring tendon autograft maturation following anatomical anterior cruciate ligament reconstruction.

PURPOSE: The primary purpose of this study was to evaluate the second-look arthroscopic findings 1 year postoperatively and magnetic resonance imaging (MRI) findings 2 years after anterior cruciate ligament reconstruction (ACLR) using bone-patellar tendon-bone autograft (BTB) or hamstring tendon autograft (HT). Secondary purpose included clinical results from physical examination, including range of motion, Lachman test, pivot shift test, and knee anterior laxity evaluation, and the clinical score for subjective evaluations at 2 years after surgery. METHODS: Between 2015 and 2018, 75 patients with primary ACL injuries were divided into either the BTB group (n = 30) or HT group (n = 45). When using HT, an anatomical double-bundle ACLR was performed. BTB was indicated for athletes with sufficient motivation to return to sporting activity. Graft maturation on second-look arthroscopy was scored in terms of synovial coverage and revascularization. All participants underwent postoperative MRI evaluation 2 years postoperatively. The signal intensity (SI) characteristics of the reconstructed graft were evaluated using oblique axial proton density-weighted MR imaging (PDWI) perpendicular to the grafts. The signal/noise quotient (SNQ) was calculated to quantitatively determine the normalized SI. For clinical evaluation, the Lachman test, pivot shift test, KT-2000 evaluation, Lysholm score, and Knee injury and Osteoarthritis Outcome Score (KOOS) were used. RESULTS: Arthroscopic findings showed that the graft maturation score in the BTB group (3.6 ± 0.7) was significantly greater than that in the anteromedial bundle (AMB; 2.9 ± 0.2, p = 0.02) and posterolateral bundle (PLB; 2.0 ± 0.9, p = 0.001) in the HT group. The mean MRI-SNQs were as follows: BTB, 2.3 ± 0.5; AMB, 2.9 ± 0.9; and PLB, 4.1 ± 1.1. There were significant differences between BTB, AMB, and PLB (BTB and AMB: p = 0.04, BTB and PLB: p = 0.003, AMB and PLB: p = 0.03). Second-look arthroscopic maturation score and MRI-SNQ value significantly correlated for BTB, AMB, and PLB. No significant differences were detected in clinical scores. There was a significant difference (p = 0.02) in the knee laxity evaluation (BTB: 0.9 ± 1.1 mm; HT: 2.0 ± 1.9 mm). CONCLUSION: BTB maturation is superior to that of double-bundle HT based on morphological and MRI evaluations following anatomical ACLR, although no significant differences were found in clinical scores. Regarding clinical relevance, the advantages of BTB may help clinicians decide on using the autograft option for athletes with higher motivation to return to sporting activity because significant differences were observed in morphological evaluation, MRI assessment, and knee anterior laxity evaluation between BTB and double-bundle HT. LEVEL OF EVIDENCE: Level IV.

[A Case of Advanced Adenocarcinoma at the Esophagogastric Junction That Exhibited pCR by Preoperative SOX Therapy].

Although neoadjuvant chemotherapy(NAC)is an effective treatment option for advanced adenocarcinoma at the esophagogastric junction (AEG), there is no sufficient evidence of this in Japan. We report a case of advanced AEG with pathological complete response(pCR)after NAC with S-1 and oxaliplatin(SOX). A 39-year-old man was diagnosed with advanced AEG cT3(SS)N0M0, cStage ⅡB. A total of 3 courses of SOX was administered. After the chemotherapy, the primary tumor showed a significant reduction in size. Subsequently, laparoscopic proximal gastrectomy, D1+ lymphadenectomy and double-flap technique reconstruction were performed. Histopathological examinations showed no residual cancer cells in the resected specimen. Thus, preoperative SOX therapy can be one of the useful treatment strategies for advanced AEG.

Joint effusion at 6 months is a significant predictor of joint effusion 1 year after anterior cruciate ligament reconstruction.

PURPOSE: This study aimed to assess the risk factors for prolonged joint effusion in patients undergoing double-bundle anterior cruciate ligament reconstruction (ACLR). METHODS: In total, 160 patients who underwent primary ACLR using autograft hamstring between 2015 and 2018 were retrospectively reviewed. Joint effusion was defined as any grade ≥ 2 (range, 0-3) according to the MRI Osteoarthritis Knee Score (MOAKS). Univariate and multivariate logistic regression analyses were performed. RESULTS: The median age of the patients was 25 years (range 14-68 years) at the time of the surgery; there were 89 women and 71 men. At 1 year, 46 (28.8%) patients experienced knee joint effusion, as defined by the MOAKS. Univariate analysis revealed that age, preoperative Kellgren-Lawrence (K-L) grade, and joint effusion at 6 months were significantly associated with joint effusion at 1 year. In the multivariate analysis, joint effusion at 6 months was significantly associated with joint effusion at 1 year (odds ratio, 68.0; 95% confidence interval, 22.1-209.4). No significant difference in the Lysholm scores was observed between patients with and without joint effusion at 1 year (n.s.). CONCLUSIONS: Joint effusion at 6 months was significantly associated with joint effusion 1 year after ACLR. LEVEL OF EVIDENCE: III.

Femoral nerve versus adductor canal block for early postoperative pain control and knee function after anterior cruciate ligament reconstruction with hamstring autografts: a prospective single-blind randomised controlled trial.

INTRODUCTION: The optimal pain management strategy for postoperative pain after anterior cruciate ligament reconstruction (ACLR) remains unclear. This study compared femoral nerve block (FNB) and adductor canal block (ACB) for pain management of early postoperative pain, knee function, and recovery of activity of daily living (ADL) after ACLR using hamstring autografts. MATERIAL AND METHODS: In this prospective, single-blind, randomised controlled trial, 64 patients aged 12-56 years who underwent anatomical double-bundle ACLR with a hamstring autograft between August 2019 and May 2020 were randomised to undergo preoperative FNB (n = 32) or ACB (n = 32). The peripheral nerve block was performed by a single experienced anaesthesiologist under ultrasound guidance. The primary outcomes were postoperative pain as evaluated using the visual analogue scale (VAS) at 3, 6, 12, 24, and 48 h postoperatively and the need for pain relief. The secondary outcome was knee function, including the recovery of range of motion, contraction of the vastus medialis, and stable walking with a double-crutch (ADL), as evaluated by blinded physical therapists. RESULTS: There were no significant differences in patient demographics between the two groups. The VAS scores, need for pain relief, knee function, and ADL did not significantly differ between the groups. CONCLUSION: FNB and ACB provided comparable outcomes related to early postoperative pain, knee function, and ADL after double-bundle ACLR using hamstring autografts. Further research is necessary to evaluate the mid- to long-term effect of each block on recovery of knee function and ADL. LEVEL OF EVIDENCE: I.

GGC Repeat Expansion of NOTCH2NLC in Adult Patients with Leukoencephalopathy.

Leukoencephalopathies comprise a broad spectrum of disorders, but the genetic background of adult leukoencephalopathies has rarely been assessed. In this study, we analyzed 101 Japanese patients with genetically unresolved adult leukoencephalopathy using whole-exome sequencing and repeat-primed polymerase chain reaction for detecting GGC expansion in NOTCH2NLC. NOTCH2NLC was recently identified as the cause of neuronal intranuclear inclusion disease. We found 12 patients with GGC expansion in NOTCH2NLC as the most frequent cause of adult leukoencephalopathy followed by NOTCH3 variants in our cohort. Furthermore, we found 1 case with de novo GGC expansion, which might explain the underlying pathogenesis of sporadic cases. ANN NEUROL 2019;86:962-968.

[Trajectory of evacuees' health condition after the Great East Japan Earthquake: Health survey of residents in prefabricated temporary housing and private rented housing in the Miyagi prefecture].

Objectives The Great East Japan Earthquake occurred in March 2011. As of November 2018, 1100 survivors of its still lived in the Miyagi prefecture's temporary housing. Previous studies revealed that the residential relocation to temporary housing from their own houses due to damages caused by the earthquake exacerbated the evacuees' health. However, there is a lack of long-term observation of the trajectory of their health conditions in temporary housing. To the best of our knowledge, there is no study involving residents in public disaster housing. The aim of the present study was to reveal the trajectory of health conditions of residents of temporary and public disaster housing.Method This repeated cross-sectional study included residents aged 20 years and older, residing in private rented housing, prefabricated temporary housing, and public disaster housing. The survey period lasted for six years (2011 to 2017). We used self-rated health as the dependent variable, the year of the survey and type of house as independent variables, and sex and age as covariates. Adjusted odds ratios (aORs) and 95% confidence intervals (95% CIs) were calculated using multivariate logistic regression analysis.Results In total, 179,255 participants were included in this study. The average age was the highest among the residents of public disaster housing: 63.0 years in 2017. The number of residents who reported poor self-rated health declined yearly of private rented housing, but not of prefabricated temporary housing. The highest number of residents reporting poor self-rated health was from public disaster housing, compared to other temporary housing. Multivariate analysis showed that self-rated health improved in recent years (P for trend <0.001). Compared to the residents of private rented housing, those from public disaster housing reported poorer health conditions (aOR, 1.20 ; 95% CI, 1.15-1.27), although no significant difference was observed among the residents of prefabricated housing.Conclusion Health conditions of residents of temporary housing and public disaster housing tended to improve on a yearly basis. Residents of public disaster housing reported poor health. Therefore, monitoring and adequate intervention should be offered.

Visible-light driven hydrogen production using chlorophyll derivatives conjugated with a viologen moiety in the presence of platinum nanoparticles.

The utilization of the light-harvesting and electron-transferring function of chlorophylls (Chls) has received attention for visible-light driven hydrogen production. In this work, a series of Chl derivatives based on pyropheophorbide-a (Pyro-a) conjugated with a viologen moiety, including a Pyro-a methyl ester directly bonded with the viologen at the 3-position 1, its 31-methylene analog 2 and Pyro-a connected with the viologen in the 17-substituent 3, were synthesized from chemical modification of naturally occurring Chl-a and characterized in terms of their photochemical and photophysical properties. As the photoexcited singlet state of the Pyro-a moiety was strongly quenched by the viologen moiety in a molecule, the effective photoinduced intramolecular electron transfer from Pyro-a to the bonded viologen moiety occurred. Moreover, these molecules were applied as a photosensitizer in the system for visible-light driven hydrogen production with platinum nanoparticles via intramolecular reduction of the bonded viologen moiety. Efficient photoreduction of external methyl viologen and successive hydrogen production on platinum nanoparticles were achieved using the synthetic conjugate of Pyro-a with the viologen moiety as a photosensitizer. In particular, effective visible-light driven hydrogen production was accomplished using 3 and platinum nanoparticles via the reduction of external methyl viologen.

C31-Selective substitution of cationic N-heteroaromatic groups into a 3-vinylated chlorophyll-a derivative.

A variety of N-containing heteroarenes were site-selectively introduced at the C31-position of a chlorophyll-a derivative possessing a 3-vinyl group through a C-N+ bond via oxidative reactions using iodine in the presence of a silver(i) salt. Electron-rich N-heteroarenes were effectively substituted, while electron-withdrawing or bulky nitrogen-neighboring substituents suppressed the reactivities. The cationic products formed were characterized by 1D/2D NMR, and their optical properties were also investigated.

Inferior graft maturity in the PL bundle after autograft hamstring double-bundle ACL reconstruction.

PURPOSE: The purpose of this study was to evaluate the signal/noise quotient (SNQ) for graft maturation and the serial changes observed in the magnetic resonance imaging (MRI) findings after double-bundle (DB) anterior cruciate ligament (ACL) reconstruction using a hamstring tendon autograft at a minimum of 5 years after surgery. METHODS: Forty-five patients who underwent DB ACL reconstruction between 2007 and 2010 were included in this prospective study. All participants underwent postoperative MRI at 3 weeks and 3, 6, 9 and 12, 18, 24, 36, 48 and 50 months. The signal intensity (SI) characteristics of the reconstructed graft were evaluated on oblique axial proton density-weighted MR imaging (PDWI) perpendicular to the grafts. The signal/noise quotient (SNQ) was calculated to quantitatively determine the normalized SI. The SNQ of the AMB and PLB was evaluated separately. RESULTS: The mean SNQ of the AM bundle (AMB) continued to increase until 6 months after surgery (5.2 ± 1.2), and then gradually decreased and became well stabilized by 18 months (3.3 ± 0.5), after which it remained unchanged. On the other hand, the mean SNQ of the PL bundle (PLB) continued to increase until 9 months after surgery (6.2 ± 1.1), and then decreased incrementally and became well stabilized by 24 months (4.1 ± 0.5). The SI of PLB was significantly higher than that of AMB between 3 and 24 months (p = 0.04, 0.03, 0.01, 0.04, 0.02 and 0.03, respectively). CONCLUSIONS: These results indicate that at least 18 months is needed after ACL reconstruction to sufficiently restore the SI of the AMB, while at least 24 months are needed to for the PLB. The SI of the PLB was significantly higher than that of the AMB at 3-24 months after surgery, indicating that the PLB showed inferior graft maturity to the AMB until 24 months after surgery. For clinical relevance, the correct understanding of serial changes in graft maturation may potentially be used in decision-making regarding a return to sports. LEVEL OF EVIDENCE: Prospective case series, Level IV.

A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration.

Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.

Characterization of a novel allergenic protein from the octocoral Scleronephthya gracillima (Kuekenthal) that corresponds to a new GFP-like family named Akane.

Certain marine organisms have been known to cause allergic reactions among occupational fishermen. We have previously reported that bronchial asthma among the workers engaged in spiny lobster fishing in Japan was caused by octocorals such as Dendronephthya sp. and Scleronephthya gracillima (previously named Alcyonium gracillimum). Now we have found another octocoral, Scleronephthya gracillima (Kuekenthal), which causes the allergic disease in fishermen. The octocoral was characterized as a new green fluorescent protein (GFP)-like family. The new allergen has a molecular mass of 27 kDa in 1D and 2D SDS-PAGE under reduced conditions. The 27 kDa component was determined to be an allergen by western blotting, ECL immune staining method and absorption of patient sera with the antigen. Furthermore, the combination of analysis with LC-ESI-MS/MS and MASCOT search in the NCBInr database concluded the 27 kDa component had the sequence YPADI/LPDYFK, and that the 22 kDa component had the sequence QSFPEGFSWER, which both matched a GFP-like protein in Acropora aculeus and in Montastraea annularis. Further analysis by MALDI-TOF/MS/MS and MASCOT search in the NCBInr database of all 27 kDa eight spot components from 2D SDS-PAGE indicated that the sequence QSFPEGFSWER also matched as GFP-like protein in Lobophyllia hemprichii and Scleractinia sp. To our knowledge, this is the first report of the new allergenic protein that corresponds to a new GFP-like protein named Akane, and which has fluorescent emissions in the red and green part of the spectra at 628 nm and 508 nm, respectively.

Increasing Incidence of Tuberculosis Infection in the Coastal Region of Northern Miyagi after the Great East Japan Earthquake.

On March 11, 2011, the Great East Japan Earthquake struck off the northeast coast of Japan. Within an hour of the earthquake, devastating tsunamis swept over the coastal region of the Miyagi Prefecture, facing Pacific Ocean. Accordingly, more than 400,000 residents were forced to stay at evacuation shelters. We investigated the changes in tuberculosis prevalence after the disaster. Annual data for all tuberculosis patients between April 1, 2009 and March 31, 2013 were extracted from the database of the Miyagi Prefectural Government. In the coastal region of Northern Miyagi, the number of tuberculosis patients increased in the post-disaster period (p < 0.001, 9.6 vs.19.1 per 100,000 people), compared to the pre-disaster period. In contrast, its prevalence did not change in the inland region of Northern Miyagi and the coastal and inland regions of Southern Miyagi. Importantly, in the inland and coastal regions of Northern Miyagi, the number of patients with latent tuberculosis infection (LTBI) increased in the post-disaster period (p < 0.001). Furthermore, in the coastal shelters, 11 evacuees with the history of contacting tuberculosis patients were diagnosed with LTBI, whereas no cases of LTBI patients were observed in the inland shelters. Thus, staying in the coastal shelters was a risk factor for contracting tuberculosis (OR: 19.31, 95% CI: 1.11-334.80); indeed, twice as many evacuees visited each coastal shelter on April 1, 2011, compared to the inland region. We should prepare the shelters to avoid overcrowding, and long-term observation is required to detect the prevalence of tuberculosis infection.

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin.

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.

Development of a novel immunoassay specific for mouse intact proinsulin.

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM.

Isolated Unilateral Ptosis due to Paramedian Midbrain Infarction.

A 59-year-old man who had hypertension, dyslipidemia, diabetes mellitus, and left eye glaucoma developed sudden vertigo and left ptosis; he did not notice diplopia. He visited our hospital on day 3 after onset and neurologic examination showed left ptosis. His left visual acuity was counting fingers, and the light reflex was sluggish owing to glaucoma. Pupil sizes were equal, and eye movements and the lower lid were unremarkable. Magnetic resonance images revealed an acute infarction of the left paramedian midbrain. We considered that selective damage to the oculomotor fascicles innervating the left levator palpebrae superioris caused ipsilateral ptosis. As the fascicles for this ocular muscle run in the small area adjacent to those for the medial rectus, inferior rectus and superior rectus muscles, this is an extremely rare case of midbrain infarction presenting with isolated unilateral ptosis.