Neuromodulation with transcranial direct current stimulation contributes to motor function recovery via microglia in spinal cord injury.

Spinal cord injury (SCI) is damage or trauma to the spinal cord, which often results in loss of function, sensation, or mobility below the injury site. Transcranial direct current stimulation (tDCS) is a non-invasive and affordable brain stimulation technique used to modulate neuronal circuits, which changes the morphology and activity of microglia in the cerebral cortex. However, whether similar morphological changes can be observed in the spinal cord remains unclear. Therefore, we evaluated neuronal population activity in layer 5 (L5) of M1 following SCI and investigated whether changes in the activities of L5 neurons affect microglia-axon interactions using C57BL/6J mice. We discovered that L5 of the primary motor cortex (corticospinal neurons) exhibited reduced synchronized activity after SCI that correlates with microglial morphology, which was recovered using tDCS. This indicates that tDCS promotes changes in the morphological properties and recovery of microglia after SCI. Combining immunotherapy with tDCS may be effective in treating SCI.

Longitudinal impact of multi-segment spinal fixation surgery on mobility status and clinical outcomes in adult spinal deformity: a multicenter retrospective study.

PURPOSE: To investigate changes in postoperative mobility status in patients with ASD, and the determining factors that influence these changes and their impact on clinical outcomes, including the rate of home discharge and long-term mobility. METHODS: A total of 299 patients with ASD who underwent multi-segment posterior spinal fusion were registered in a multi-center database were investigated. Patient mobility status was assessed using walking aids and classified into five levels (1: independent, 2: cane, 3: walker, 4: assisted, and 5: wheelchair) preoperatively, at discharge, and after 2 years. We determined improvements or declines in the patient's mobility based on changes in the classification levels. The analysis focused on the factors contributing to the deterioration of postoperative mobility. RESULTS: Two years postoperatively, 87% of patients maintained or improved mobility. However, 27% showed decreased mobility status at discharge, associated with a lower rate of home discharge (49% vs. 80% in the maintained mobility group) and limited improvement in mobility status (35% vs. 5%) after 2 years. Notably, postoperative increases in thoracic kyphosis (7.0 ± 12.1 vs. 2.0 ± 12.4°, p = 0.002) and lower lumbar lordosis (4.2 ± 13.1 vs. 1.8 ± 12.6°, p = 0.050) were substantial factors in mobility decline. CONCLUSION: Postoperative mobility often temporarily decreases but generally improves after 2 years. However, an overcorrection in sagittal alignment, evidenced by increased TK, could detrimentally affect patients' mobility status. Transient mobility decline associated with overcorrection may require further rehabilitation or hospitalization. Further studies are required to determine the biomechanical effects of surgical correction on mobility.

Does global spinal alignment affect the use of walking aids after multi-segment spinal fixation for patients with ASD? A multicenter retrospective study.

PURPOSE: This study aimed to clarify the relation between global spinal alignment and the necessity of walking aid use in patients with adult spinal deformity (ASD) and to investigate the impact of spinal fixation on mobility status after surgery. METHODS: In total, 456 older patients with ASD who had multi-segment spinal fixation surgery and were registered in a multi-center database were investigated. Patients under 60 years of age and those unable to walk preoperatively were excluded. Patients were classified by their mobility status into the independent, cane, and walker groups. Comparison analysis was conducted using radiographic spinopelvic parameters and the previously reported global spine balance (GSB) classification. In addition, preoperative and 2 years postoperative mobility statuses were investigated. RESULTS: Of 261 patients analyzed, 66 used walking aids (canes, 46; walkers, 20). Analysis of preoperative radiographical parameters showed increased pelvic incidence and pelvic incidence-lumbar lordosis mismatch in the walker group and increased sagittal vertebral axis in the cane and walker groups versus the independent group. Analysis of GSB classification showed a higher percentage of walker use in those with severe imbalance (grade 3) in the sagittal classification but not in the coronal classification. While postoperative radiographical improvements were noted, there was no significant difference in the use of walking aids before and 2 years after surgery (P = 0.085). CONCLUSION: A significant correlation was found between "sagittal" spinal imbalance and increased reliance on walking aids, particularly walkers. However, the limitation of improvement in postoperative mobility status suggested that multiple factors influence the mobility ability of elderly patients with ASD.

Preoperative low Hounsfield units in the lumbar spine are associated with postoperative mechanical complications in adult spinal deformity.

PURPOSE: To determine the most valid bone health parameter to predict mechanical complications (MCs) following surgery for adult spinal deformity (ASD). METHODS: This multicenter study retrospectively examined the records of patients who had undergone fusion of three or more motion segments, including the pelvis, with a minimum two-year follow-up period. Patients with moderate and severe global alignment and proportion scores were included in the study and divided into two groups: those who developed MCs and those who did not. Bone mineral density (BMD) of the lumbar spine and femoral neck was measured using dual-energy X-ray absorptiometry, and Hounsfield units (HUs) were measured in the lumbar spine on computed tomography. Radiographic parameters were evaluated preoperatively, immediately after surgery, and at final follow-up. RESULTS: Of 108 patients, 30 (27.8%) developed MCs, including 26 cases of proximal junctional kyphosis/failure, 2 of distal junctional failure, 6 of rod fracture, and 11 reoperations. HUs were significantly lower in patients who experienced MCs (113.7 ± 41.1) than in those who did not (137.0 ± 46.8; P = 0.02). BMD did not differ significantly between the two groups. The preoperative and two-year postoperative global tilt, as well as the immediately postoperative sagittal vertical axis, were significantly greater in patients who developed MCs than in those who did not (P = 0.02, P < 0.01, and P = 0.01, respectively). CONCLUSION: Patients who experienced MCs following surgery for ASD had lower HUs than those who did not. HUs may therefore be more useful than BMD for predicting MCs following surgery for ASD.

Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens.

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Modulation of the Pharmacokinetics of a Radioligand Targeting Carbonic Anhydrase-IX with Albumin-Binding Moieties.

The expression of carbonic anhydrase-IX (CA-IX) in tumors can lead to a poor prognosis; thus, CA-IX has attracted much attention as a target molecule for cancer diagnosis and treatment. An 111In-labeled imidazothiadiazole sulfonamide (IS) derivative, [111In]In-DO3A-IS1, exhibited marked tumor accumulation but also marked renal accumulation, raising concerns about it producing a low signal/background ratio and a high radiation burden on the kidneys. In this study, four 111In-labeled IS derivatives, IS-[111In]In-DO2A-ALB1-4, which contained four different kinds of albumin binder (ALB) moieties, were designed and synthesized with the aim of improving the pharmacokinetics of [111In]In-DO3A-IS1. Their utility for imaging tumors that strongly express CA-IX was evaluated in mice. An in vitro binding assay of cells that strongly expressed CA-IX (HT-29 cells) was performed using acetazolamide as a competitor against CA-IX, and IS-[111In]In-DO2A-ALB1-4 did not exhibit reduced binding to HT-29 cells compared with [111In]In-DO3A-IS1. In contrast, IS-[111In]In-DO2A-ALB1-4 showed a greater ability to bind to human serum albumin than [111In]In-DO3A-IS1 in vitro. In an in vivo biodistribution study, the introduction of an ALB moiety into the 111In-labeled IS derivative markedly decreased renal accumulation and increased HT-29 tumor accumulation and blood retention. The pharmacokinetics of the IS derivatives varied depending on the substituted group within the ALB moiety. Single-photon emission computed tomography imaging with IS-[111In]In-DO2A-ALB1, which showed the highest tumor/kidney ratio in the biodistribution study, facilitated clear HT-29 tumor imaging, and no strong signals were observed in the normal organs. These results indicate that IS-[111In]In-DO2A-ALB1 may be an effective CA-IX imaging probe and that the introduction of ALB moieties may improve the pharmacokinetics of CA-IX ligands.

Synthesis and evaluation of 68Ga-labeled imidazothiadiazole sulfonamide derivatives for PET imaging of carbonic anhydrase-IX.

INTRODUCTION: Carbonic anhydrase-IX (CA-IX) is markedly overexpressed in many types of solid tumors promoting tumorigenicity and tumor growth. We synthesized novel 68Ga-labeled imidazothiadiazole sulfonamide (IS) derivatives ([68Ga]Ga-DO3A-IS1 and [68Ga]Ga-DO2A-IS2), and evaluated their utility as positron emission tomography (PET) probes targeting CA-IX. METHODS: [67/68Ga]Ga-DO3A-IS1 and [67/68Ga]Ga-DO2A-IS2 were synthesized from corresponding precursors by ligand substitution reaction in acetate buffer. Cell binding assays were performed using HT-29 cells, which highly express CA-IX, and MDA-MB-231 cells, which show lower-level expression of CA-IX, and a biodistribution assay with model mice bearing the HT-29 or MDA-MB-231 tumor was performed. [68Ga]Ga-DO3A-IS1 was further evaluated by PET/CT. RESULTS: To evaluate their fundamental properties, [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 were synthesized by conjugation with 67Ga, which has a much longer decay half-life and can be utilized more easily than 68Ga. [67/68Ga]Ga-DO3A-IS1 and [67/68Ga]Ga-DO2A-IS2 were prepared from corresponding precursors with preferable yield and purity. [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 showed significantly greater binding to HT-29 cells than MDA-MB-231 cells in vitro and the binding of [67Ga]Ga-DO2A-IS2 to HT-29 cells was much greater than that of [67Ga]Ga-DO3A-IS1, suggesting multivalent interactions. [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 showed significant selectivity for the HT-29 tumor in vivo, while tumor uptake of [67Ga]Ga-DO3A-IS1 was greater than that of [67Ga]Ga-DO2A-IS2. PET/CT of [68Ga]Ga-DO3A-IS1 showed selectivity for the HT-29 tumor, although [68Ga]Ga-DO3A-IS1 could not be used to visualize the HT-29 tumor clearly because of its strong background signals. CONCLUSION: These results indicate that 68Ga-labeled IS derivatives may be useful 68Ga-PET probes targeting CA-IX with further structural modifications.

Synthesis and evaluation of indium-111-labeled imidazothiadiazole sulfonamide derivative for single photon emission computed tomography imaging targeting carbonic anhydrase-IX.

Carbonic anhydrase-IX (CA-IX) is a zinc enzyme overexpressed in the hypoxic regions of many types of solid tumors; therefore, in vivo imaging of CA-IX may contribute to cancer diagnosis. In this study, we newly designed and synthesized an 111In-labeled CA-IX imaging agent based on an imidazothiadiazole sulfonamide (IS) scaffold conjugated with a chelating moiety, DO3A ([111In]DO3A-IS1), and evaluated its utility for imaging of CA-IX high-expressing tumors. [111In]DO3A-IS1 was successfully synthesized at a 76% radiochemical yield by reacting its precursor with 111InCl3 in acetate buffer. In in vitro assays, [111In]DO3A-IS1 showed marked stability in murine plasma and greater binding to CA-IX high-expressing (HT-29) cells (118 ± 21% initial dose/mg protein) than CA-IX low-expressing (MDA-MB-231) cells (1.4 ± 0.3% initial dose/mg protein). Moreover, in an in vivo biodistribution assay, [111In]DO3A-IS1 showed marked accumulation in the HT-29 tumor (8.71 ± 1.41% injected dose/g at 24 h postinjection). In addition, in a single photon emission computed tomography (SPECT) study, [111In]DO3A-IS1 clearly and selectively visualized the HT-29 tumor as compared with the MDA-MB-231 tumor. These results indicate that [111In]DO3A-IS1 may serve as a useful SPECT imaging agent with the novel scaffold targeting CA-IX.

Molecular epidemiology of Clostridioides difficile and risk factors for the detection of toxin gene-positive strains.

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients' backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18-18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery.

Burrow Morphology of Alpheid Shrimps: Case Study of Alpheus brevicristatus and a Review of the Genus.

The Alpheus brevicristatus De Haan, 1844 is one of the commonest shrimp species inhabiting the tidal flats in Japan. This species is sometimes accompanied by the facultative symbiotic goby, Acentrogobius spp. Here, we investigated the burrow morphology of A. brevicristatus in a tidal flat of Uranouchi Inlet, Kochi Prefecture, Japan. We also reviewed existing literature on alpheid burrow morphology using the resin casting technique, to determine how burrows vary in the presence and absence of gobies. Nine burrows were casted in situ using polyester resin. All burrows were long, but shallow in structure, with several funnel-shaped openings and short cul-de-sac branches. This species appears to use several burrow openings to access the sediment surface for feeding with high efficiency. Gobies were not associated with all burrows cast; however, 1-3 individuals of the small alpheid shrimp Athanas japonicus Kubo, 1936 were entombed in seven of the casts. A review of 12 studies on the burrow morphology of 16 Alpheus species based on resin casting techniques showed wide variation in burrow characteristics, such as burrow depth, length, and number of openings. Our findings suggest that burrow structure is influenced by species-specific characteristics and sediment type. The possibility that the presence of the symbiotic goby affects the burrow morphology of Alpheus shrimp is discussed.

Strong influence of human leukocyte antigen-DP variants on response to hepatitis B vaccine in a Japanese population.

Genome-wide association studies (GWASs) have reported that human leukocyte antigen (HLA) variants are associated with chronic hepatitis B, spontaneous hepatitis B virus (HBV) clearance, and response to hepatitis B vaccine. Single nucleotide polymorphisms (SNPs) in HLA-DP (rs9277535 and rs3077) and HLA-DQ (rs2856718 and rs7453920) have been repeatedly associated with chronic hepatitis B and spontaneous HBV clearance. However, the data on the SNPs associated with response to hepatitis B vaccine are inconclusive. The objective of this study was to determine whether these four HLA SNPs that have been identified as risk loci for chronic HBV infection are associated with response to hepatitis B vaccine in a Japanese population. We enrolled 278 medical students who received hepatitis B vaccination and measured anti-hepatitis B surface (HBs) antibody titers 1month after a three-dose vaccination series. We found that rs9277535 and rs3077 in HLA-DP were strongly associated with response to hepatitis B vaccine (odds ratio [OR]=0.31 and 0.32, P=0.004 and 0.010, respectively). These two SNPs were significantly associated with anti-HBs titers in an allele-dependent manner. On the other hand, rs2856718 and rs7453920 in HLA-DQ were not associated with response to hepatitis B vaccine. These results indicate that rs9277535 and rs3077 in HLA-DP are the major determinants of response to hepatitis B vaccine, whereas rs2856718 and rs7453920 in HLA-DQ have little effect on the immune response to hepatitis B vaccine.

Performance evaluation of the Verigene®Clostridium difficile nucleic acid test, an automated multiplex molecular testing system for detection of C. difficile toxin.

The Verigene®Clostridium difficile nucleic acid test (Verigene® CDF test) is an automatic and rapid detection system for the genes encoding tcdA, tcdB, binary toxin, and the single nucleotide deletion at base pair 117 in the tcdC based on microarray and PCR amplification. We compared the performance of the Verigene® CDF test to that of two enzyme immunoassays, C. DIFF QUIK CHEK COMPLETE and X/Pect Toxin A/B, using 118 specimens. We found overall concordance rates of 81.4% and 78.8% between C. DIFF QUIK CHEK COMPLETE and Verigene® CDF test, and X/Pect Toxin A/B and Verigene® CDF test. The Verigene® CDF test showed the highest sensitivity (93.9%) and had a specificity of 96.5%. The sensitivity and specificity were respectively 45.5 and 94.1% for C. DIFF QUIK CHEK COMPLETE and 27.3 and 100.0% for X/Pect Toxin A/B. These results indicated that the Verigene® CDF test was highly accurate for the detection of C. difficile toxin in fecal specimens and supported its use in daily diagnostic practice.

Improved assay for differential diagnosis between Pompe disease and acid α-glucosidase pseudodeficiency on dried blood spots.

The high frequency (3.3-3.9%) of acid α-glucosidase pseudodeficiency, c.[1726G>A; 2065G>A] homozygote (AA homozygote), in Asian populations complicates newborn screening for Pompe disease (glycogen storage disease type II or acid maltase deficiency) on dried blood spots, since AA homozygotes have a considerably low enzyme activity. We observed that hemoglobin in the enzyme reaction solution strongly interferes with the fluorescence of 4-methylumbelliferone released from 4-methylumbelliferyl α-D-glucopyranoside (4MU-αGlc) by acid α-glucosidase. Therefore, we have searched for a method to effectively eliminate hemoglobin in the reaction solution. Hemoglobin precipitation with barium hydroxide and zinc sulfate (Ba/Zn method) carried out after the enzyme reaction considerably enhances the fluorescence intensity while it does not reduce the intensity to any extent as can occur with conventional deproteinization agents like trichloroacetic acid. The Ba/Zn method greatly improved the separation between 18 Japanese patients with Pompe disease and 70 unaffected AA homozygotes in a population of Japanese newborns in the assay with 4MU-αGlc on dried blood spots. No overlap was observed between both groups. We further examined acid α-glucosidase activity in fibroblasts from 11 Japanese patients and 57 Japanese unaffected individuals including 31 c.[1726G; 2065G] homozygotes, 18 c.[1726G; 2065G]/[1726A; 2065A] heterozygotes and 8 AA homozygotes to confirm that fibroblasts can be used for definitive diagnosis. The patients were reliably distinguished from three control groups. These data provide advanced information for the development of a simple and reliable newborn screening program with dried blood spots for Pompe disease in Asian populations.