Detection of typical indigenous gut bacteria related to kanpyo Lagenaria siceraria var. hispida powder in murine caecum and human faecal cultures.

Kanpyo (KP) is an edible dried product produced by peeling the fruit of the gourd Lagenaria siceraria var. hispida; it is used in the traditional Japanese cuisine. The health functionality of KP due to its rich dietary fibre is expected to include a possible combined effect of KP-responsive indigenous gut bacteria (KP-RIB). However, its effect on the gut microbiota is unclear. To determine the effects of the KP on the gut microbiota and their host, Institute of Cancer Research mice were fed a high-sucrose diet containing no fibre (NF) or 5% (w/w) KP for 14 days, and their caecal microbiota was analysed by 16S rRNA (V4) amplicon sequencing. Higher faecal frequency and weight and lower spleen weight and spleen tumour necrosis factor-α levels were observed in KP-fed mice than in NF-fed mice (p < 0.05). KP increased and decreased the abundance of short-chain fatty acid producer Lachnospiraceae and obesity-inflammation related Allobaculum species, respectively. In the case of human faecal cultures, stool samples from five healthy volunteers were inoculated and incubated at 37 °C for 24 h anaerobically; 3.2% (w/v) KP suppressed putrefactive compounds (indole, phenol, and ammonia). KP increased butyrate-producer Faecalibacterium, acetate/lactate-producer Bifidobacterium, and Lachnospira. Furthermore, KP cultures showed high antioxidant and RAW264.7 macrophage cell activation capacities. These results suggest that KP-RIB and KP intake may synergistically affect host health. However, further studies are required to clarify the synergistic effects of KP and KP-RIB.

Maximum knee extension velocity without external load is a stronger determinant of gait function than quadriceps strength in the early postoperative period following total knee arthroplasty.

OBJECTIVE: Quadriceps weakness is considered the primary determinant of gait function after total knee arthroplasty (TKA). However, many patients have shown a gap in improvement trends between gait function and quadriceps strength in clinical situations. Factors other than quadriceps strength in the recovery of gait function after TKA may be essential factors. Because muscle power is a more influential determinant of gait function than muscle strength, the maximum knee extension velocity without external load may be a critical parameter of gait function in patients with TKA. This study aimed to identify the importance of knee extension velocity in determining the gait function early after TKA by comparing the quadriceps strength. METHODS: This prospective observational study was conducted in four acute care hospitals. Patients scheduled for unilateral TKA were recruited (n = 186; age, 75.9 ± 6.6 years; 43 males and 143 females). Knee extension velocity was defined as the angular velocity of knee extension without external load as quickly as possible in a seated position. Bilateral knee function (knee extension velocity and quadriceps strength), lateral knee function (pain and range of motion), and gait function (gait speed and Timed Up and Go test (TUG)) were evaluated before and at 2 and 3 weeks after TKA. RESULTS: Both bilateral knee extension velocities and bilateral quadriceps strengths were significantly correlated with gait function. The knee extension velocity on the operation side was the strongest predictor of gait function at all time points in multiple regression analysis. CONCLUSION: These findings identified knee extension velocity on the operation side to be a more influential determinant of gait function than impairments in quadriceps strength. Therefore, training that focuses on knee extension velocity may be recommended as part of the rehabilitation program in the early postoperative period following TKA. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR) UMIN000020036.

Lactoferrin-like Immunoreactivity in Distinct Neuronal Populations in the Mouse Central Nervous System.

Lactoferrin (Lf) is an iron-binding glycoprotein mainly found in exocrine secretions and the secondary granules of neutrophils. In the central nervous system (CNS), expression of the Lf protein has been reported in the lesions of some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as in the aged brain. Lf is primarily considered an iron chelator, protecting cells from potentially toxic iron or iron-requiring microorganisms. Other biological functions of Lf include immunomodulation and transcriptional regulation. However, the roles of Lf in the CNS have yet to be fully clarified. In this study, we raised an antiserum against mouse Lf and investigated the immunohistochemical localization of Lf-like immunoreactivity (Lf-LI) throughout the CNS of adult mice. Lf-LI was found in some neuronal populations throughout the CNS. Intense labeling was found in neurons in the olfactory systems, hypothalamic nuclei, entorhinal cortex, and a variety of brainstem nuclei. This study provides detailed information on the Lf-LI distribution in the CNS, and the findings should promote further understanding of both the physiological and pathological significance of Lf in the CNS.

Short-Term Efficacy and Safety of Omidenepag Isopropyl in Patients with Normal-Tension Glaucoma.

PURPOSE: To retrospectively evaluate the short-term efficacy of omidenepag isopropyl (EYBELIS 0.002%) by assessing its intraocular pressure (IOP)-lowering capability and safety in patients with normal-tension glaucoma (NTG). PATIENTS AND METHODS: Fifty-four NTG patients (54 eyes) who were newly administrated with omidenepag isopropyl were enrolled in the study. The subjects comprised 22 men and 32 women, and the mean age of the subjects was 55.0 ± 14.1 years. The mean deviation value using the Humphrey visual field test program (30-2 SITA Standard) was -5.03 ± 3.38 dB. The following data were retrieved from the medical records and used for retrospective analyses: IOP at baseline 1-2 months and 3-4 months after administration. The frequency of non-responder patients who had less than 10% IOP reduction was evaluated. Patients were observed for adverse reactions and dropouts at each time point. RESULTS: IOP at baseline, after 1-2 months and after 3-4 months was 15.7 ± 2.6 mmHg, 13.5 ± 2.3 mmHg, and 13.6 ± 2.4 mmHg, respectively. There was a significant decrease in IOP after administration (p<0.0001). Eleven patients (22.4%) were non-responders. Adverse reactions occurred in 4 patients (7.4%), including conjunctival hyperemia in 3 patients (after 1 week, 2 weeks, and 1 month, respectively) and eye pain in 1 patient (after 1 month). Five patients (9.3%) dropped out of the study because of an adverse reaction in 3 patients, insufficient IOP reduction in 1 patient, and discontinuation of follow-up of 1 patient at our institution. CONCLUSION: After administration of omidenepag isopropyl, IOP in patients with NTG was significantly decreased. However, adverse reactions occurred in 7.4% of patients.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

Preliminary study on the effects of movement velocity training of the upper limbs on gait ability in older adults: a nonrandomized controlled trial.

Purpose: Movement velocity of the limbs or trunk plays an important determinant of gait speed in older adults. Movement velocity-focused training of the lower limbs or trunk has recently been shown to be an effective intervention to improve gait ability. Because movement velocities of various body regions are significantly correlated, movement velocity training of the upper limbs may also be effective for improving gait speed. Therefore, the purpose of this study was to investigate whether movement velocity training of the upper limbs in a seated position is effective for improving gait ability. Patients and methods: This study was a nonrandomized controlled trial. The participants were older adults residing in geriatric health service facilities. They were assigned to the movement velocity training of the upper limbs group (n=26) or control group (n=15). The participants in the training group performed exercises (three times per week for 10 weeks) to move the upper limbs as quickly as possible. The outcomes were gait speed, movement velocity, and quadriceps strength. These measurements were performed preintervention and 4, 8, and 10 weeks after intervention. Results: A significant time-group interaction was found for maximum gait speed and movement velocity of the upper limbs. Bonferroni post-hoc test showed significant improvement in gait speed between preintervention and 10 weeks after intervention in the training group. The movement velocity of the upper limbs was significantly improved between preintervention and 4, 8, and 10 weeks after intervention. Conclusion: Movement velocity training of the upper limbs showed significant and clinically relevant improvements in maximum gait speed at 10 weeks after intervention. This training is a potentially useful intervention and can be safely performed.

Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a rapid gene-targeting technology that does not require embryonic stem cells. To demonstrate dosage effects of the Pax6 gene on eye formation, we generated Pax6-deficient mice with the CRISPR/Cas system. Eyes of founder embryos at embryonic day (E) 16.5 were examined and categorized according to macroscopic phenotype as class 1 (small eye with distinct pigmentation), class 2 (pigmentation without eye globes), or class 3 (no pigmentation and no eyes). Histologically, class 1 eyes were abnormally small in size with lens still attached to the cornea at E16.5. Class 2 eyes had no lens and distorted convoluted retinas. Class 3 eyes had only rudimentary optic vesicle-like tissues or histological anophthalmia. Genotyping of neck tissue cells from the founder embryos revealed somatic mosaicism and allelic complexity for Pax6. Relationships between eye phenotype and genotype were developed. The present results demonstrated that development of the lens from the surface ectoderm requires a higher gene dose of Pax6 than development of the retina from the optic vesicle. We further anticipate that mice with somatic mosaicism in a targeted gene generated by CRISPR/Cas-mediated genome editing will give some insights for understanding the complexity in human congenital diseases that occur in mosaic form.

Expression analysis of Dickkopf-related protein 3 (Dkk3) suggests its pleiotropic roles for a secretory glycoprotein in adult mouse.

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.

Beneficial effects of protease preparations derived from Aspergillus on the colonic luminal environment in rats consuming a high-fat diet.

The present study investigated the effects of the dietary addition of the protease preparations derived from Aspergillus on the colonic luminal environment. Rats were fed a 30% beef tallow diet with or without the protease preparations, including Amano protease (protease A 'Amano SD', neutral proteases from Aspergillus spp.) or orientase (orientase AY, acid proteases from Aspergillus niger) at the dose of 0.2% for 3 weeks. Cecal Bifidobacterium was significantly elevated in the dietary Amano protease group (194-fold, P<0.05), but not in the orientase group. Lactobacillus was elevated in the two groups (P<0.05). Cecal n-butyrate, propionate and lactate were higher in the Amano protease and orientase groups compared with the controls (P<0.05). Fecal immunoglobulin A and mucins were elevated in the Amano protease group (P<0.05). These results suggest the potential effect of the consumption of Aspergillus-derived protease preparations that are favorable for the colonic luminal environment in rats fed a high-fat diet.

Cecal succinate elevated by some dietary polyphenols may inhibit colon cancer cell proliferation and angiogenesis.

This study demonstrated that 0.5% dietary rutin, ellagic acid, or curcumin markedly increased cecal succinate levels in rats fed a high-fat diet, whereas catechin, caffeic acid, and quercetin did not. Other organic acids were modestly or hardly affected by polyphenols. To clarify the effects of succinate levels increased by polyphenols, this study examined the effects of succinate on the growth and proliferation of colon cancer cells and angiogenesis. The growth and proliferation of HT29 human colon cancer cells and angiogenesis in an ex vivo model were significantly inhibited by succinate at a dose close to that in the cecum of rats fed polyphenols. Furthermore, succinate inhibited the migration of human umbilical vein endothelial cells. These findings suggest that the consumption of some polyphenols affects the health and diseases of the large intestine by elevating succinate.

The expression of LIM-homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation.

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye-related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM-homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle-like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.

Burdock fermented by Aspergillus awamori elevates cecal Bifidobacterium, and reduces fecal deoxycholic acid and adipose tissue weight in rats fed a high-fat diet.

This study investigated the effects of dietary supplementation with burdock powder and Aspergillus awamori-fermented burdock powder at 5% on the intestinal luminal environment and body fat in rats fed a high-fat (HF) diet. Food intake and growth were unaffected by dietary manipulation. Consumption of the burdock and fermented burdock diets significantly elevated fecal IgA and mucins (indices of intestinal immune and barrier functions) and reduced fecal lithocholic acid (a risk factor for colon cancer) (p<0.05). The fermented burdock diet markedly elevated cecal Bifidobacterium and organic acids, including lactate, acetate, propionate, and butyrate, and reduced fecal deoxycholic acid (a risk factor for colon cancer) and perirenal adipose tissue weight (p<0.05), but the burdock diet did not. These results suggest that consumption of fermented burdock improves the intestinal luminal environment and suppresses obesity in rats fed a HF diet.

Electrokinetic supercharging preconcentration prior to CGE analysis of DNA: sensitivity depends on buffer viscosity and electrode configuration.

Aiming to high sensitivity DNA analysis by CGE, electrokinetic supercharging (EKS) approach was adopted in this article. EKS is known as an online preconcentration technique that combines electrokinetic sample injection (EKI) with transient ITP (tITP). Herein, two factors of buffer viscosity and electrode configuration were studied to further improve EKS performance. An ultralow-viscosity Tris-Boric acid-EDTA (TBE) buffer solution, consisted of 2% low-molecular-weight hydroxypropyl methyl cellulose (HPMC) and 6% mannitol and with pH 8.0 adjusted by boric acid, was applied. The boric acid would make a complex with mannitol and generates borate polyanion, which acts as the leading ion for tITP process. The new electrode configuration, a Pt ring around capillary, was modified on Agilent CE system to lead large amount sample introduction during EKS. The standard DNA sample of φX174/HaeIII digest was used to evaluate the qualitative and quantitative abilities of the proposed strategy. The 170,000-fold highly diluted sample at concentration of 3.0 ng/mL was enriched by EKS and detected by normal UV detection method. The obtained LOD of the weakest peak of 72 bp fragment was around 7.7 pg/mL, apparently improved more than 10,000-fold in comparison with conventional CGE with UV detection.

Lhx1 in the proximal region of the optic vesicle permits neural retina development in the chicken.

How the eye forms has been one of the fundamental issues in developmental biology. The retinal anlage first appears as the optic vesicle (OV) evaginating from the forebrain. Subsequently, its distal portion invaginates to form the two-walled optic cup, which develops into the outer pigmented and inner neurosensory layers of the retina. Recent work has shown that this optic-cup morphogenesis proceeds as a self-organizing activity without any extrinsic molecules. However, intrinsic factors that regulate this process have not been elucidated. Here we show that a LIM-homeobox gene, Lhx1, normally expressed in the proximal region of the nascent OV, induces a second neurosensory retina formation from the outer pigmented retina when overexpressed in the chicken OV. Lhx2, another LIM-homeobox gene supposed to be involved in early OV formation, could not substitute this function of Lhx1, while Lhx5, closely related to Lhx1, could replace it. Conversely, knockdown of Lhx1 expression by RNA interference resulted in the formation of a small or pigmented vesicle. These results suggest that the proximal region demarcated by Lhx1 expression permits OV development, eventually dividing the two retinal domains.

Sheath-flow electrochemical detection of amino acids with a copper wire electrode in capillary electrophoresis.

Here, we report the detection of native amino acids using a sheath-flow electrochemical detector with a working electrode made of copper wire. A separation capillary that was inserted into a platinum tube in the detector acted as a grounded electrode for electrophoresis and as a flow channel for sheath liquid. Sheath liquid flowed outside the capillary to support the transport of the separated analytes to the working electrode for electrochemical detection. The copper wire electrode was aligned at the outlet of the capillary in a wall-jet configuration. Amino acids injected into the capillary were separated following elution from the end of the capillary and detection by the copper electrode. Three kinds of copper electrodes with different diameters-50, 125, and 300 µm-were examined to investigate the effect of the electrode diameter on sensitivity. The peak widths of the analytes were independent of the diameter of the working electrode, while the 300-µm electrode led to a decrease in the signal-to-noise ratio compared with the 50- and 125-µm electrodes, which showed no significant difference. The flow rate of the sheath liquid was also varied to optimize the detection conditions. The limits of detection for amino acids ranged from 4.4 to 27 µM under optimal conditions.

Intravitreal injection of autologous plasmin enzyme for macular edema associated with branch retinal vein occlusion.

PURPOSE: To investigate the effects of an intravitreal injection of autologous plasmin enzyme for macular edema associated with a branch retinal vein occlusion (BRVO). DESIGN: Prospective, interventional case studies. METHODS: Twenty-six eyes of 26 patients (14 men and 12 women) with macular edema resulting from a BRVO were studied. None of the eyes had a posterior vitreous detachment (PVD). The average age at the onset of BRVO was 58.7 ± 6.6 years. The interval between onset of the macular edema and injection of autologous plasmin enzyme was 4.2 months. One international unit of autologous plasmin enzyme in 0.1 mL was injected into each vitreous. The presence of a PVD was evaluated 1 week after the injection, and the visual acuity and macular thickness were measured before and 1 week and 1, 3, 6, and 12 months after the autologous plasmin enzyme injection. RESULTS: After the autologous plasmin enzyme injection, a total PVD was observed in 23 eyes and no PVD was observed in 3 eyes. The visual acuity gradually recovered after the injection, improved by 2 lines or more in 23 eyes, and remained unchanged in the 3 eyes. The retinal thickness also recovered gradually at 1, 3, 6, and 12 months. The mean retinal thickness was reduced from 602.12 to 253.62 µm at 12 months after surgery. It was reduced significantly after 1, 3, 6, and 12 months (P < .0001). CONCLUSIONS: Intravitreal autologous plasmin enzyme may lead to an improvement of visual acuity and a reduction of macula edema in eyes with BRVO.

Consumption of some polyphenols reduces fecal deoxycholic acid and lithocholic acid, the secondary bile acids of risk factors of colon cancer.

This study was performed to examine the effect of dietary polyphenols on fecal secondary bile acids, such as deoxycholic acid and lithocholic acid, the risk factors of colon cancer, in rats fed a high-fat diet. In experiment 1, rats were fed a 30% beef tallow diet containing 0.5% polyphenols for 3 weeks. Dietary curcumin and caffeic acid significantly reduced the fecal concentration of deoxycholic acid. Dietary caffeic acid, catechin, rutin, and ellagic acid significantly reduced fecal lithocholic acid. Fecal hyodeoxycholic acid, a metabolite of lithocholic acid, was markedly lowered by dietary curcumin, caffeic acid, catechin, and rutin. In experiment 2, rats were fed a 30 or 5% beef tallow diet with or without the addition of 0.5% curcumin. In the rats without receiving curcumin, the fecal level of deoxycholic acid was significantly higher in the high-fat diet group than in the low-fat diet group. Fecal deoxycholic acid was significantly reduced by dietary curcumin in the high-fat diets but not in the low-fat diets. The results suggest novel effects of some polyphenols favorable for colon health by reducing secondary bile acids in animals fed a high-fat diet.

Two cases of severe degeneration of the macula following vitrectomy with indocyanine green-assisted internal limiting membrane peeling for idiopathic macular hole.

We describe three eyes of two cases of severe degeneration of the macula following vitrectomy with indocyanine green-assisted internal limiting membrane peeling for idiopathic macular hole. We need to remember the possibility of these complications and have to select the procedures that are safest to use for macular hole surgery.

A sheath-flow electrochemical detector for capillary electrophoresis.

In this study, a novel electrochemical detection system for capillary electrophoresis was proposed. In the proposed system, sheath flow would transport analytes to the working electrode surface to allow electrochemical detection. The sheath-flow electrochemical detector would require no modification of capillaries and could accommodate capillaries larger than 25 microm i.d.