Novel analgesics targeting brain-derived neurotrophic factor for neuropathic pain

　Brain-derived neurotrophic factor (BDNF) is necessary for the development, growth, and maintenance of nerve cells. BDNF is expressed in the dorsal root ganglion (DRG); binds to the Tropomyosin receptor kinasa B (TrkB) receptor, which has a tyrosine kinase domain, in the spinal cord; and plays an important role as a pain modulator. BDNF expression is increased in various types of pain, including acute pain, neuropathic pain, and cancer pain. Activation of the BDNF–TrkB pathway transmits pain information. In order to inhibit the BDNF–TrkB pathway, by sequestering BDNF, we constructed a cDNA expression plasmid encoding the extracellular region of rat TrkB fused to enhanced green fluorescent protein (EGFP). When the expression plasmid vector was administered to rat models of neuropathic pain, induced by spinal nerve ligation, statistically significant relief of pain was observed in terms of a 50% paw-withdrawal threshold using the von Frey test. The expression of TrkB-EGFP mRNA was detected in L5 lumbar vertebral nerves by quantitative reverse transcriptase polymerase chain reaction. To verify the pain-suppressive effect of the expression vector, truncated TrkB protein, without EGFP, was purified, and administered to pain model rats. A statistically significant suppressive effect of the truncated TrkB protein on neuropathic pain was observed 2 days after administration. The pain-suppressive effect of the truncated TrkB protein was more effective than that of the TrkB-Fc chimera protein and lasted longer than that of the TrkB antagonist ANA-12. Our results suggested that the truncated TrkB cDNA expression vector and truncated TrkB protein could be used as molecular targeted drugs in patients with neuropathic pain

Development of a novel analgesic for cancer pain targeting brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is necessary for nerve growth. BDNF is expressed in the dorsal root ganglion (DRG) and modulates pain transduction from peripheral nociceptors. TrkB, which is a BDNF receptor with a tyrosine kinase domain, acts as a pain modulator on the cell membrane of second neuron. If an exogenous truncated TrkB lacking a tyrosine kinase domain can competitively block the binding of BDNF to endogenous TrkB, inhibitory effects on pain are expected. We constructed two expression vectors coding truncated TrkB-GFP fusion proteins, lacking intracellular tyrosine kinase domain, with and without the transmembrane domain. By transfection of the vectors to HEK293 cells, the expression and localization of the modified receptor proteins were confirmed. The truncated TrkB with the transmembrane domain, TM (+), was localized on cell membrane surface of the transfected cells, and capable of BDNF binding on cell surface. TM (-) without the transmembrane domain was secreted from the transfected cells, and the secreted TrkB protein was confirmed the capability for binding with BDNF by pull-down assay. Furthermore, we developed a rat model of cancerous osteocopic pain for evaluating an analgesic effect of the modified TrkB vectors on cancer pain. Pain-related behavior, as assessed by von Frey tests, indicated hyperalgesia after cancer cell administration. BDNF expression was higher on the affected side of the DRG at the third lumbar vertebra L3 than on the unaffected side. When the modified TrkB vectors were administrated to the cancer pain model rats, both the TM (+) and TM (-) vector administration groups exhibited an analgesic effect. These results suggest that the modified TrkB receptors and their vectors are applicable as molecular targeted drugs for pain control in cancer patients

Pathological analysis of spermatic dysfunction following testicular ischemia-reperfusion injury\n

　Introduction & Objectives: Torsion, which may result in testicular ischemia, requires emergency surgery to restore testicular blood flow. However, the risk of spermatic dysfunction remains even if surgery is performed. The pathology of spermatic dysfunction in testicular ischemia-reperfusion injury (TIRI) remains unclear. A previous study showed the relevance of inflammation and oxidative stress in the other organs of ischemia-reperfusion injury. We hypothesized that inflammation and oxidative stress play key roles in causing spermatic dysfunction following TIRI. We investigated the pathophysiology of spermatic dysfunction in TIRI focusing on inflammatory changes using TIRI model mice.　Materials and Methods: The study used C57BL/6J male mice aged 10 to 15 weeks. To create TIRI model mice, the unilateral (left side) testicular vessels were clamped using Dieffenbach clamps (Bulldog clamps) for 1 hour and de-clamped. The bilateral testes were removed at 0 (ischemic state), 1, 3, and 5 weeks after creating the TIRI model mice. Spermatic changes following TIRI were investigated by analyzing the histology of the testes and semen and assessing levels of inflammation and oxidative stress. Semen was collected from the bilateral cauda epididymites and investigated using the sperm motility analysis system (SMAS).　Results: Histological analysis after hematoxylin-eosin staining showed tissue thickening in interstitial tissues at week 1 and 3 on the left (affected) testis, and week 1, 3 and 5 on the right (unaffected) testis. The infiltration of lymphocytes-predominant inflammatory cells were observed at week 1 and week 3 on the left (affected) testis. The destruction of ductal structures and giant cells were observed at weeks 3 and 5 on the left (affected) testis and week 5 on the right (unaffected) testis. SMAS showed significantly decreased spermatic concentration and motility in both testes of TIRI model mice compared with those of sham-operated mice at weeks 1, 3 and 5. Inflammation analysis using an inflammation-related proteome assay showed significantly increased levels of cytokines (IL-2, IL-3, IL-17A, and IL-23) and chemokines (CCL2, CCL5, CXCL1, and CX3CL1) at weeks 1, 3, and 5 in both testes of TIRI model mice. For the assessment of oxidative stress, enzyme-linked immuno-sorbent assay (ELISA) for 8-hydroxy-2’-deoxyguanosine (8-OHdG) was performed, which showed that levels of 8-OHdG were significantly increased in the left (affected) testis of TIRI model mice compared with that of sham-operated mice at all observation periods. Meanwhile, ELISA showed that levels of 8-OHdG in the right (unaffected) testis were significantly increased in TIRI model mice at weeks 3 and 5 compared with that of sham-operated mice.　Conclusions: Spermatic dysfunction following TIRI is induced by inflammation and oxidative stress. Inflammation and oxidative stress may be novel regulatory factors to prevent spermatic dysfunction following TIRI

Pathophysiological analysis of detrusor overactivity following partial bladder outlet obstruction

　Introduction: Detrusor overactivity (DO) following partial bladder outlet obstruction (PBOO) is a common urological condition in humans, with 50-70% patients with PBOO complicated with DO. The pathological mechanisms of DO following PBOO are largely unknown, but inflammatory changes may play a key role. We hypothesized that inflammation is important in the earlier pathophysiological phase before overproduction of oxidative stress in DO following PBOO. Therefore, we investigated the relationships among bladder function, ischemia, oxidative stress and inflammation in DO following PBOO in PBOO model mice.　Materials and Methods: C57BL/6J male mice aged 10 to 15 weeks were used in the study. PBOO model mice were created surgically by ligation of the proximal urethra with 5-0 nylon suture under inhalation anesthesia. Sham-operated mice were used as controls. Pathophysiological changes in the bladder at 1, 3 and 5 weeks after creation of the PBOO model mice were compared with those in sham-operated mice using functional, histological, biochemical and immunohistochemical analyses.　Results: Functional analysis using a pressure flow study showed increased maximum detrusor pressure at 1 week and DO from 3 to 5 weeks after creation of the PBOO model. Histological analysis using hematoxylin-eosin and Masson-Trichrome staining showed greater invasion of inflammatory cells and fibrosis in PBOO model mice compared with sham-operated mice at 3 and 5 weeks. Inflammatory cells were mainly present in interstitial tissue, and fibrosis gradually infiltrated from interstitial tissue to the muscular layer. Ischemia analysis showed significantlyincreased HIF-1α in PBOO model mice at all time points. Oxidative stress analysis indicated significantly increased levels of ROS from 1 week and 8-OHdG from 3weeks in PBOO model mice. An inflammation-related proteome assay showed high levels of colony stimulating factor (CSF) family proteins at 1 week and IL-2, IL-3, IL-17A, IL-23, MMP-3, MMP-9 and periostin from 3 to 5 weeks in PBOO model mice.　Conclusions: Oxidative stress and inflammatory changes showed contemporaneous increase in pathophysiology of detrusor overactivity following partial bladder outlet obstruction. Especially, CSF family and ROS changes are showed in the early stage, and might be a predict marker in the pathophysiology of DO following PBOO at the early stage

Preventive effect of indoleamine 2,3-dioxygenase 1 inhibition on lipopolysaccharide-induced prostatitis

　Introduction and Objectives: Bacterial infections are the main cause of acute prostatitis and are treated with appropriate antimicrobial therapy. However, approximately 5% of patients continue to have inflammatory symptoms even after receiving antibacterial therapy, leading to refractory conditions. Bacterial prostatitis requires additional therapy, focusing on inflammatory changes. Indoleamine 2,3-dioxygenase 1 (IDO1) catalysis is the first rate-limiting step of tryptophan metabolism. IDO1 is expressed in the prostate and plays a key role in the immune response. As the first step in investigating the relationship between acute prostatitis and IDO1, we investigated the preventive effect of IDO1 inhibition on lipopolysaccharide (LPS)- induced prostatitis using IDO knockout (Ido1 −/−) mice in this study.　Materials and Methods: The study used Ido1 −/− and wild-type (Ido1 +/+) C57BL/6J malemice aged 10–15 weeks. LPS Escherichia coli O26 (100μg/PBS, 100μL) was administered transurethrally into the lower urinary tract to create a mouse model of LPS-induced prostatitis. The prostates were removed 1, 3, 5, and 7 days after creating the model mice. Histological, immunohistochemical, and biochemical analyses were used to compare the preventive effect in Ido1 −/− mice compared with that in Ido1+/+ mice.　Results: HE staining showed suppression of ductal destruction following infiltration of inflammatory cells in Ido1 −/− mice compared with Ido1 +/+ mice. The enzyme-linked immunosorbent assay (ELISA) method was used for kynurenine pathway analysis, which showed significantly maintained tryptophan levels and decreased L-kynurenine levels in Ido1 −/− mice compared to Ido1 +/+ mice. The IDO1 assay in Ido1 +/+ mice showed significantly increased levels during all observation periods after creating the model compared with that under normal conditions. Immunofluorescent staining using five types of cytokines and chemokines (IL-2, IL-4, IL-17, CCL2, and CCL3) related to the pathophysiology of acute prostatitis showed decreased expression of these cytokines and chemokines in Ido1 -/- mice compared with Ido1 +/+ mice. Inflammation-related proteome assays showed decreased levels of IL-1β, IL-4, IL-5, IL-6, IL-17, CCL2, CCL3, CXCL1, CXCL11, and tissue inhibitor of matrix metalloproteinases (TIMP)-1 in Ido1 −/− mice compared with Ido1 −/− mice during all observation periods after model creation.　Conclusions: IDO1 is involved in LPS-induced prostatitis through cytokines and chemokines. IDO1 inhibition contributes to the prevention of LPS-induced prostatitis. IDO1 inhibition has the potential to serve as an additional therapy for acute prostatitis

Changes in serum antibody titers after vaccination for COVID-19 and evaluation of post-vaccination health conditions

　Introduction: The coronavirus disease 2019 (COVID-19) vaccine has preventive effects and high immunogenicity, but the outcomes of vaccination have not been widely reported. The goal of this study was to examine serum antibody titers before and after vaccination and to evaluate post-vaccination health conditions.　Methods: The subjects were 2,304 medical workers (mean age 41 years) at Kawasaki Gakuen who agreed to participate in the study and underwent COVID-19 vaccination, beginning in March 2021. Serum IgG antibody titers for SARS-CoV-2 spike protein were measured before the first vaccination and 4 weeks after the second vaccination. Health conditions were observed for 4 weeks after the second vaccination.　Results: The rates of seroconversion, seroprotection, and change in geometric mean antibody titer at 4 weeks after the second vaccination were 99.9%, 99.9%, and 2,685.5 (95% CI 587.8-5,319.2), respectively, suggesting high immunogenicity. After the first vaccination, pain, enlargement, and reddening occurred at the local injection site, and systemic side effects included fatigue, headache, physical pain, chill, nausea, and fever. After the second vaccination, the incidence of pain decreased, but those of other events increased. There were no serious side effects requiring hospitalization. In logistic regression analysis, sex, age, fever,chill, and lymph node enlargement after the second vaccination were associated with a change in antibody titer.　Conclusions: Serum antibody titers suggested high immunogenicity of the COVID-19 vaccine and a health condition survey confirmed the safety of the vaccine. Systemic side effects may serve as an index of immunization (acquisition of antibody) by the vaccine

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Web application to convert English into helpful characters for pronunciation learners

The International Phonetic Alphabet (IPA) is the most commonly used set of phonetic symbols but it can be difficult to understand and too abstract for non-phoneticians, such as English learners and foreign language educators. One of the factors that makes it difficult for English learners is the number of vowels used in English. In previous research, this problem was solved by proposing a condensed list of 13 English vowels and 24 consonants that are logically organized for North American English, and by assigning a new phonetic symbol font called Sound Spelling to these phonemes. However, there are currently no English texts that have Sound Spelling to show learners this easy way to pronounce and it is difficult for non-phoneticians to write transcriptions right away, because materials using those symbols are lacking for English learners. In this research, we developed a web application, now publicly available, that converts input English into Sound Spelling quickly and accurately to solve this problem

Complexity of Tiling a Polygon with Trominoes or Bars

We study the computational hardness of the tiling puzzle with polyominoes, where a polyomino is a rectilinear polygon (i.e., a polygon made by connecting unit squares.) In the tiling problem, we are given a rectilinear polygon P and a set S of polyominoes, and asked whether P can be covered without any overlap using translated copies of polyominoes in S. In this paper, we focus on trominoes and bars as polyominoes; a tromino is a polyomino consisting of three unit squares, and a bar is a rectangle of either height one or width one. Notice that there are essentially two shapes of trominoes, that is, I-shape (i.e., a bar) and L-shape. We consider the tiling problem when restricted to only L-shape trominoes, only I-shape trominoes, both L-shape and I-shape trominoes, or only two bars. In this paper, we prove that the tiling problem remains NP-complete even for such restricted sets of polyominoes. All reductions are carefully designed so that we can also prove the #P-completeness and ASP-completeness of the counting and the another-solution-problem variants, respectively.Our results answer two open questions proposed by Moore and Robson (2001) and Pak and Yang (2013)