Exoskeleton rehabilitation robot training for balance and lower limb function in sub-acute stroke patients: a pilot, randomized controlled trial.

PURPOSE: This pilot study aimed to investigate the effects of REX exoskeleton rehabilitation robot training on the balance and lower limb function in patients with sub-acute stroke. METHODS: This was a pilot, single-blind, randomized controlled trial. Twenty-four patients with sub-acute stroke (with the course of disease ranging from 3 weeks to 3 months) were randomized into two groups, including a robot group and a control group. Patients in control group received upright bed rehabilitation (n = 12) and those in robot group received exoskeleton rehabilitation robot training (n = 12). The frequency of training in both groups was once a day (60 min each) for 5 days a week for a total of 4 weeks. Besides, the two groups were evaluated before, 2 weeks after and 4 weeks after the intervention, respectively. The primary assessment index was the Berg Balance Scale (BBS), whereas the secondary assessment indexes included the Fugl-Meyer Lower Extremity Motor Function Scale (FMA-LE), the Posture Assessment Scale for Stroke Patients (PASS), the Activities of Daily Living Scale (Modified Barthel Index, MBI), the Tecnobody Balance Tester, and lower extremity muscle surface electromyography (sEMG). RESULTS: The robot group showed significant improvements (P < 0.05) in the primary efficacy index BBS, as well as the secondary efficacy indexes PASS, FMA-LE, MBI, Tecnobody Balance Tester, and sEMG of the lower limb muscles. Besides, there were a significant differences in BBS, PASS, static eye-opening area or dynamic stability limit evaluation indexes between the robotic and control groups (P < 0.05). CONCLUSIONS: This is the first study to investigate the effectiveness of the REX exoskeleton rehabilitation robot in the rehabilitation of patients with stroke. According to our results, the REX exoskeleton rehabilitation robot demonstrated superior potential efficacy in promoting the early recovery of balance and motor functions in patients with sub-acute stroke. Future large-scale randomized controlled studies and follow-up assessments are needed to validate the current findings. CLINICAL TRIALS REGISTRATION: URL: https://www.chictr.org.cn/index.html.Unique identifier: ChiCTR2300068398.

MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion.

Mitochondria are double-membraned organelles with variable shapes influenced by metabolic conditions, developmental stage, and environmental stimuli. Their dynamic morphology is a result of regulated and balanced fusion and fission processes. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and the maintenance of membrane potential. Mitofusins are dynamin-related GTPases that are essential for mitochondrial fusion. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via combined oligomerization and GTP hydrolysis. However, the molecular mechanisms of this process remain unknown. Here we present crystal structures of engineered human MFN1 containing the GTPase domain and a helical domain during different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of MFN1 and resembles the 'neck' of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote GTPase domain dimerization in the transition state. Disruption of GTPase domain dimerization abolishes the fusogenic activity of MFN1. Moreover, a conserved aspartate residue trigger was found to affect mitochondrial elongation in MFN1, probably through a GTP-loading-dependent domain rearrangement. Thus, we propose a mechanistic model for MFN1-mediated mitochondrial tethering, and our results shed light on the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders.

First report of stem-end rot caused by Botryosphaeria fabicerciana on mango fruits in China.

Mango (Mangifera indica L.), belongs to the family Anacardiacea, and is one of the most popular tropical fruits in the world. Stem-end rot is a major postharvest disease of mango fruit, causing severe losses during storage in China (Chen et al., 2015). In July 2021, the mango fruits harvested from Baise Municipal National Agricultural Science and Technology Park (23.683568 N, 106.986325 E) of Guangxi province in China developed stem-end rot during storage. The disease incidence reached ca. 8.3%. The initial symptoms appeared as light brown lesions surrounding the peduncle, which quickly expanded becoming large dark-brown lesions. Small pieces of epidermis (5 mm × 5 mm) from 8 typical diseased friuts were cut from the edges of lesions surface-sterilized with 2% sodium hypochlorite and rinsed with sterile distilled water. The tissue was plated on potato dextrose agar (PDA) and incubated at 28 ℃ in the dark for 3 days. Fifteen, similarcolonies were isolated from the symptomatic tissue. The representative isolates DF-1, DF-2 and DF-3 were selected for morphological characterization, molecular identification, and pathogenicity testing. The colonies were circular with fluffy aerial mycelium, initially white turning to smoke-gray from the center in upper side and greenish black in reverse side, covering the 90 mm diameter Petri dish after 4 days of incubation on PDA at 28 ℃ in dark. Pycnidia were produced on the surface of the colony after 30 days. Conidia were fusiform, aseptate, hyaline, thin-walled with granular contents, apex sub-obtuse, base subtruncate to bluntly rounded, 14.0-20.3 (16.8±1.6) µm × 3.1-7.2 (5.1±0.9) µm (n=50). The sexual stage was absent. Based on morphology, isolates were preliminarily identified as Botryosphaeria speices. To accurately identify the pathogen, genomic DNA was extracted from the mycelium of the three isolates DF-1, DF-2 and DF-3. The internal transcribed spacer of rDNA region (ITS), elongation factor 1-alpha (EF-1α) and beta-tubulin gene (TUB) genes were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b, respectively (Slippers et al., 2004). The nucleotide sequences were all deposited in GenBank (ITS: OP729176-OP729178 EF-1α: OP758194-OP758196 and TUB: OP758197-OP758199). Based on the BLASTn analysis, the ITS, EF1-α and TUB sequences of three isolates were 100%, 99% and 99% similar to the Botryosphaeria fabicerciana MFLUCC 10-0098 sequences (ITS: JX646789, EF-1α: JX646854 and TUB: JX646839). Multi-locus phylogenetic analyses (ITS, EF-1α and TUB) showed that the isolate DF-1, DF-2 and DF-3 were clustered within Botryosphaeria fabicerciana clade based on the maximum likelihood , Bayesian inference, and maximum parsimony methods. The pathogenicity test was performed by placing discs mycelium around the peduncle of mature mango fruits by pin-prick method. Each treatment carried out with 12 fruits. The inoculated fruits were placed in plastic boxes at 28 ℃ with three replicates. Three days after inoculation, typical symptoms of stem-end rot were observed. The control fruits were inoculated with sterile PDA discs, and remained symptomless. The same fungus was re-isolated from the symptomatic tissue to complete Koch's postulate. Botryosphaeria fabicerciana (basionym: Fusicoccum fabicercianum) was first reported as pathogen causing senescent twig of Eucalyptus spp. in China (Chen et al., 2011; Phillips et al., 2013). To our knowledge, this is the first report of Botryosphaeria fabicerciana causing stem-end rot of Mangifera indica in China.

First report of anthracnose caused by Colletotrichum karstii on Dalbergia odorifera in China.

Dalbergia odorifera (Family: Fabaceae) is a national second-grade protected tree in China with high medicinal and economic value (Zhao et al., 2020). In July, 2022, a leaves spot disease on D. odorifera with typical anthracnose symptoms was observed in plantations in Haikou (110.319153°E, 19.072900°N), Dongfang (108.630297°E, 19.103838°N) and Qiongzhong (109.704460°E, 19.088440°N), Hainan Province, China. Disease incidence was 7.5% (n = 50 plants). Early symptoms of infected leaves were small and round dark brown spots, which developed into larger irregular necrotic lesions and leaves withered. Leaf tissues (5×5 mm) at the disease-health junction of spots from 19 leaves were sterilized with 2.5% sodium hypochlorite for 1 min, and rinsed with sterile distilled water three times. These sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 28 ℃ for 5 d. 7 strains of fungi with similar morphology were isolated, and 3 single-hyphal isolates (HHL01, HHL02 and HHL03) from each location were selected for further study. Colonies on PDA were fluffy orange-yellow mycelium. Conidia were aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, 11.82 to 15.77 × 3.87 to 6.71 µm (n = 100; average = 13.75 × 5.52 µm). Appressoria formed on slides, measured 5.54 to 10.64 × 4.19 to 7.41 µm (n = 30; average = 8.06 × 5.97 µm) were brown to black, elliptical to irregular. For molecular biological identification, the genomic DNA of three isolates was extracted by fungal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The partial sequences of internal transcribed spacer region (ITS; ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GDF1/GDR1), actin (ACT; ACT512F/ACT783R), ß-tubulin (TUB2; TI/Bt2b) and calmodulin (CAL; CL1C/CL2C) were amplified and sequenced by Sangon Biotech (Shanghai) Co., Ltd (Carbone and Kohn, 1999; Weir et al., 2012). The sequences were deposited as GenBank Accession Nos. OR018110-OR018112 (ITS); OR050529-OR050537 (GAPDH, ACT and CAL) and OR192168-OR192170 (TUB). BLASTn results showed these sequences were more than 99% identity with the strain of C. karstii CORCK1 (GenBank Accession Nos. HM585406, HM585387, HM581991, HM585424 and HM582010, respectively). Multi-locus phylogenetic tree of Colletotrichum spp. showed that those three isolates were sister to C. karstii based on the maximum likelihood and bayesian inference methods. To verify pathogenicity, 2 mL spore suspension (1 × 106conidia/ml) of the isolates was sprayed on each leaves of 1-year-old D. odorifera plants, and sterile distilled water was similarly sprayed on other leaves as a negative control. The plants were incubated in a greenhouse under 90% ± 5% RH at 28 °C. Light brown small round necrotic patches developed 3 days after inoculation, while the control was asymptomatic. Photographs were taken on the fifth day after inoculation. The fungi were re-isolated from the diseased leaves and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. karstii has been reported causing leaf rot of Carissa grandiflora in Spain (Garcia-Lopez et al., 2021), and anthracnose caused by C.tropicale was reported on D. odorifera (Yi et al., 2023). To our knowledge, this is the first report of Dalbergia odorifera leaf spot disease caused by Colletotrichum karstii. This finding provides an important basis for further research on the control of the disease.

First report of Neofusicoccum sinoeucalypti causing leaf spot on Terminalia catappa in China.

Terminalia catappa belonging to the family Combretaceae, spreads in tropical and subtropical coastal areas. It mainly serves as shading and decorative tree (Anand et al, 2015). It is planted as roadside tree in Southern China. A leaf spot disease of T. catappa was observed at Wencheng Town (110.805323°E, 19.524567°N), Wenchang City, Hainan province, China in June, 2022. The disease incidence of leaves reached 10%. The occurrence of this leaf spot would reduce the ornamental value of T. catappa. The early symptoms of infected leaves were small, round, dark brown spots surrounded by irregular light halos, developing to larger irregular necrotic lesions and leaves withered. Twelve diseased leaves were collected from three survey trees. Symptomatic leaf samples were collected and cut into small pieces (3×3 mm). The pieces were surface sterilized with 2.5% sodium hypochlorite for 1 min, rinsed with sterile distilled water three times, placed on potato dextrose agar (PDA) medium and incubated at 28 ℃ in the dark for 3 days. Three hyphal tip isolates (DYLR-1, DYLR-2 and DYLR-3) were cultured on PDA. Colonies on PDA reached the edge of the 90 mm plates after 3 d and had fluffy mycelia with an uneven margin, initially creamy white, becoming light grey (5 d) to mouse grey (10 d) at the surface with the black globular cavity. To induce sporulation, the isolates were transferred to 2% water agar media with sterilised pine needles placed on the surface of the media. Conidia was hyaline, unicellular, thin-walled, smooth with granular contents, aseptate, narrowly fusiform, base subtruncate to bluntly rounded, 11.1 to 16.7 (14.5±1.4) × 4.6 to 7.6 (6.2±0.7) µm (n=50). Spermatia was hyaline, unicellular, aseptate, allantoid to rod-shaped, 3.2 to 6.9 (5.1±0.9) µm × 2.0 to 3.8 (2.5±0.4) µm (n=50). Pathogenicity tests were performed both in vitro and in vivo, and replicated twice. All three isolates were used for pathogenicity tests, with 18 detached leaves used for pathogenicity tests in vitro and 3 seedlings used for pathogenicity tests in vivo. A 5-mm-diameter agar plug containing mycelia were placed on the leaves both without and with wound. Sterile PDA plugs were used as controls. The leaves were moisturized with a clear plastic bag for 24 hours in a greenhouse under 90% ± 5% RH at 25 ℃. Brown spot symptoms were observed at 1 day post-inoculation (dpi) in vitro and 3 dpi in vivo. The same strains were reisolated from lesions of inoculated leaves. Control plants were symptomless. For molecular identification, internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS; ITS1/ITS4 primers; White et al. 1990), translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF1-986R primers; Carbone and Kohn 1999), beta-tubulin gene (tub2; Bt2a/Bt2b primers; Glass and Donaldson 1995) and DNA directed RNA polymerase II second largest subunit gene (rpb2; RPB2bot6F/RPB2bot7R; Sakalidis et al. 2011) regions were PCR amplified from genomic DNA. The sequences (GenBank accessions numbers: OP435357 to OP435359 of ITS; OP535354 to OP535356 of tef1-α; OP535351 to OP535353 of tub2; OP535348 to OP535350 of rpb2) had 100%, 99.7%, 100%, 100% similar to the type strain of Neofusicoccum sinoeucalypti CERC2005 (GenBank accessions numbers: KX278061, KX278166, KX278270 and KX278290), respectively. Multi-locus phylogenetic tree (ITS, tef1-α, tub2 and rpb2) of Neofusicoccum spp. (Zhang et al. 2021) showed that those three isolates were sister to N. sinoeucalypti based on the maximum likelihood and bayesian inference methods. N. sinoeucalypti was first reported pathogen causing from Eucalyptus plantations and adjacent plants in China (Li et al. 2018). To our knowledge, this is the first report of Neofusicoccum sinoeucalypti causing leaf spot disease on Terminalia catappa in China. Neofusicoccum species, commonly cause diseases in woody plants worldwide, and identification of this pathogen is important for effective disease management and control.

Occurrence of Anthracnose Caused by Colletotrichum fructicola on Ficus hirta in Southern China.

Ficus hirta Vahl. is a Moraceae plant, named for its palm-like leaves. It is a widely used traditional medicinal material with definite curative effect. At the same time, it is also a commonly used soup material among the folk in South China. In March 2022, a serious leaf spot disease with symptoms similar to anthracnose was observed on F. hirta in several plantations in Qinzhou and Zhanjiang City of China, with an incidence of 32~65%. The early symptoms of infected leaves were small, round, yellow spots that further expanded into larger, brown, irregular, necrotic lesions surrounded by dark brown edges, which eventually led to leaf wilt. Twenty symptomatic leaves were collected from three plantations with a total area of about 10 hm2. Fragments (2×2 mm) from the 20 infected leaves were surface sterilized, plated on potato dextrose agar (PDA) and incubated at 28°C. After 3 days, isolates with similar cultural morphology were obtained and three representative isolates (WZMT-1, WZMT-3 and WZMT-8) were randomly selected for following study. The colonies by single-spore purification on PDA were initially cottony, pale white and became grayish green with age. The conidia were hyaline, abundant, cylindrical, with rounded ends, 14.4~18.2 µm×4.6~6.0 µm (av. 16.2 µm×5.4 µm, n=100). Conidiogenous cells hyaline, cylindrical or ampulliform, 6.2~22.7 µm × 2.7~5.0 µm (av. 12.9 µm×3.8 µm, n=50). Appressoria were brown to dark brown, ovoid to clavate, elliptical or irregular, 7.9~13.4 µm × 5.6~9.2 µm (av. 10.6 µm×7.9 µm, n=50). The morphology of the fungus resembled Colletotrichum fructicola (Prihastuti et al. 2009). For molecular identification, the internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), actin (ACT), beta-tubulin 2 (TUB2), calmodulin (CAL), partial manganese superoxide dismutase (sod2), partial Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) (ApMat) genes were amplified from genomic DNA for the isolates using the primers described by Silva et al. (2012) and Weir et al. (2012). The sequences of the above seven loci of the three isolates (accession nos. OQ121661 to OQ121663 and OQ133400 to OQ133417) were obtained and showed over 99% identity with the existing sequences of ex-type culture ICMP 18581 of Colletotrichum fructicola (Weir et al. 2012). A multilocus phylogenetic analysis of the seven loci concatenated sequences using the maximum likelihood method revealed that the isolates belong to C. fructicola. To confirm pathogenicity, five 3-month-old potted plants were used for inoculation with each representative isolate. Tested plants were sprayed with 10 ml of a conidial suspension (1 × 108 conidia/ml) , and the controls plants were sprayed with sterile water. All the plants were incubated in a growth chamber at 26 ± 2°C with 95% relative humidity. After 10 days, typical lesions like those observed on the field plants appeared on all inoculated plants, while the control remained healthy. The same fungal pathogen was reisolated and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates. The pathogen has been reported as the causal agent of anthracnose on a wide range of plant hosts worldwide (Marquez-Zequera et al. 2018; Horfer et al. 2021; Jiang et al. 2022; Li et al. 2023). To our knowledge, this is the first report of anthracnose on F. hirta caused by C. fructicola in southern China.

Excellent Dark/Light Dual-Mode Photoresponsive Activities Based on g-C3N4/CMCh/PVA Nanocomposite Hydrogel Using Electron Beam Radiation Method.

Photocatalytic technology for inactivating bacteria in water has received much attention. In this study, we reported a dark-light dual-mode sterilized g-C3N4/chitosan/poly (vinyl alcohol) hydrogel (g-CP) prepared through freeze-thaw cycling and an in situ electron-beam radiation method. The structures and morphologies of g-CP were confirmed using Fourier infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), solid ultraviolet diffuse reflectance spectroscopy (UV-vis DRS), and Brunauer-Emmett-Teller (BET). Photocatalytic degradation experiments demonstrated that 1 wt% g-CP degraded rhodamine B (RhB) up to 65.92% in 60 min. At the same time, g-CP had good antimicrobial abilities for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) within 4 h. The shapes of g-CP were adjustable (such as bar, cylinder, and cube) and had good mechanical properties and biocompatibility. The tensile and compressive modulus of 2 wt% g-CP were 0.093 MPa and 1.61 MPa, respectively. The Cell Counting Kit-8 (CCK-8) test and Hoechst33342/PI double staining were used to prove that g-CP had good biocompatibility. It is expected to be applied to environmental sewage treatment and wound dressing in the future.

Tannic Acid-Assisted Immobilization of Copper(II), Carboxybetaine, and Argatroban on Poly(ethylene terephthalate) Mats for Synergistic Improvement of Blood Compatibility and Endothelialization.

Due to thrombosis and intimal hyperplasia, small-diameter vascular grafts have poor long-term patency. A combination strategy based on nitric oxide (NO) and anticoagulants has the potential to address those issues. In this study, poly(ethylene terephthalate) (PET) mats were prepared by electrospinning and coated with tannic acid (TA)/copper ion complexes. The chelated copper ions endowed the mats with sustained NO generation by catalytic decomposition of endogenous S-nitrosothiol. Subsequently, zwitterionic carboxybetaine acrylate (CBA) and argatroban (AG) were immobilized on the mats. The introduced AG and CBA had synergistic effects on the improvement of blood compatibility, resulting in reduced platelet adhesion and prolonged blood clotting time. The biocomposite mats selectively promoted the proliferation and migration of human umbilical vein endothelial cells while inhibiting the proliferation and migration of human umbilical arterial smooth muscle cells under physiological conditions. In addition, the prepared mats exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. Collectively, the prepared mats hold great promise as artificial small-diameter vascular grafts.

FAM46B is a prokaryotic-like cytoplasmic poly(A) polymerase essential in human embryonic stem cells.

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

Effectiveness of a Novel Contralaterally Controlled Neuromuscular Electrical Stimulation for Restoring Lower Limb Motor Performance and Activities of Daily Living in Stroke Survivors: A Randomized Controlled Trial.

Background: Contralaterally controlled neuromuscular electrical stimulation (CCNMES) is a novel electrical stimulation treatment for stroke; however, reports on the efficacy of CCNMES on lower extremity function after stroke are scarce. Objective: To compare the effects of CCNMES versus NMES on lower extremity function and activities of daily living (ADL) in subacute stroke patients. Methods: Forty-four patients with a history of subacute stroke were randomly assigned to a CCNMES group and a NMES group (n = 22 per group). Twenty-one patients in each group completed the study per protocol, with one subject lost in follow-up in each group. The CCNMES group received CCNMES to the tibialis anterior (TA) and the peroneus longus and brevis muscles to induce ankle dorsiflexion motion, whereas the NMES group received NMES. The stimulus current was a biphasic waveform with a pulse duration of 200 µs and a frequency of 60 Hz. Patients in both groups underwent five 15 min sessions of electrical stimulation per week for three weeks. Indicators of motor function and ADL were measured pre- and posttreatment, including the Fugl-Meyer assessment of the lower extremity (FMA-LE) and modified Barthel index (MBI). Surface electromyography (sEMG) assessments included average electromyography (aEMG), integrated electromyography (iEMG), and root mean square (RMS) of the paretic TA muscle. Results: Values for the FMA-LE, MBI, aEMG, iEMG, and RMS of the affected TA muscle were significantly increased in both groups after treatment (p < 0.01). Patients in the CCNMES group showed significant improvements in all the measurements compared with the NMES group after treatment. Within-group differences in all post- and pretreatment indicators were significantly greater in the CCNMES group than in the NMES group (p < 0.05). Conclusion: CCNMES improved motor function and ADL ability to a greater extent than the conventional NMES in subacute stroke patients.

Melatonin promotes in vitro maturation of vitrified-warmed mouse GV oocytes potentially by modulating MAD2 protein expression of SAC component through MTRs.

Previous studies have shown that melatonin (MT) can ameliorate vitrification-inflicted damage in mouse germinal vesicle (GV) oocytes, however, the key mechanistic basis of this improvement still remains poorly understood. This study was conducted to investigate whether MT can improve in vitro developmental potential of vitrified-warmed GV oocytes through its receptors. The fresh oocytes were randomly divided into four groups: untreated (control group, F), vitrified by open-pulled straw method (vitrification group, V), vitrification group with 100 nmol/L MT supplementation (vitrification + MT group, VM), and with 100 nmol/L MT plus 100 nmol/L luzindole administration (vitrification + MT + luzindole group, VML) or with 50 nmol/L ramelteon addition (vitrification + ramelteon group; VR). After warming, oocytes were cultured in vitro, and MT receptors (MTRs), MAD2 (mitotic arrest deficient 2), Securin and CyclinB1 protein levels and spindle morphology were evaluated. The ratio of oocytes developed to the metaphase I (MI) and metaphase II (MII) stages was also assessed. The results showed that after vitrification-warming, the in vitro maturation rate of GV oocytes was significantly lower compared to the control (F) group. Vitrification also significantly impaired the spindle morphology, decreased the protein level of MTRs and Securin, and decreased MAD2 levels in MI oocytes. However, when MT or ramelteon (MTRs agonist) were added (group wise) to warming and maturation media, the maturation rate of GV oocytes was significantly increased, the normal proportion of the spindle morphology increased, and the expression level of MAD2 increased in their resulting MI oocytes compared to the vitrification group. However, following addition of both MT and ramelteon, the maturation rate of GV oocyte showed no significant difference between VML and vitrification groups. The spindle morphology and MAD2 levels in MI oocytes were comparable to the vitrification group but differed significantly from the VM group. Taken together, finding of the present study shows that MT (100 nmol/L) can ameliorate the in vitro maturation of vitrified-warmed mouse GV oocytes, potentially by improving the spindle morphology, modulating MAD2 protein level and promoting the development of MI stage oocytes through MTRs.

Prediction of regulatory motifs from human Chip-sequencing data using a deep learning framework.

The identification of transcription factor binding sites and cis-regulatory motifs is a frontier whereupon the rules governing protein-DNA binding are being revealed. Here, we developed a new method (DEep Sequence and Shape mOtif or DESSO) for cis-regulatory motif prediction using deep neural networks and the binomial distribution model. DESSO outperformed existing tools, including DeepBind, in predicting motifs in 690 human ENCODE ChIP-sequencing datasets. Furthermore, the deep-learning framework of DESSO expanded motif discovery beyond the state-of-the-art by allowing the identification of known and new protein-protein-DNA tethering interactions in human transcription factors (TFs). Specifically, 61 putative tethering interactions were identified among the 100 TFs expressed in the K562 cell line. In this work, the power of DESSO was further expanded by integrating the detection of DNA shape features. We found that shape information has strong predictive power for TF-DNA binding and provides new putative shape motif information for human TFs. Thus, DESSO improves in the identification and structural analysis of TF binding sites, by integrating the complexities of DNA binding into a deep-learning framework.

An algorithmic perspective of de novo cis-regulatory motif finding based on ChIP-seq data.

Transcription factors are proteins that bind to specific DNA sequences and play important roles in controlling the expression levels of their target genes. Hence, prediction of transcription factor binding sites (TFBSs) provides a solid foundation for inferring gene regulatory mechanisms and building regulatory networks for a genome. Chromatin immunoprecipitation sequencing (ChIP-seq) technology can generate large-scale experimental data for such protein-DNA interactions, providing an unprecedented opportunity to identify TFBSs (a.k.a. cis-regulatory motifs). The bottleneck, however, is the lack of robust mathematical models, as well as efficient computational methods for TFBS prediction to make effective use of massive ChIP-seq data sets in the public domain. The purpose of this study is to review existing motif-finding methods for ChIP-seq data from an algorithmic perspective and provide new computational insight into this field. The state-of-the-art methods were shown through summarizing eight representative motif-finding algorithms along with corresponding challenges, and introducing some important relative functions according to specific biological demands, including discriminative motif finding and cofactor motifs analysis. Finally, potential directions and plans for ChIP-seq-based motif-finding tools were showcased in support of future algorithm development.

Bioinformatics tools for quantitative and functional metagenome and metatranscriptome data analysis in microbes.

Metagenomic and metatranscriptomic sequencing approaches are more frequently being used to link microbiota to important diseases and ecological changes. Many analyses have been used to compare the taxonomic and functional profiles of microbiota across habitats or individuals. While a large portion of metagenomic analyses focus on species-level profiling, some studies use strain-level metagenomic analyses to investigate the relationship between specific strains and certain circumstances. Metatranscriptomic analysis provides another important insight into activities of genes by examining gene expression levels of microbiota. Hence, combining metagenomic and metatranscriptomic analyses will help understand the activity or enrichment of a given gene set, such as drug-resistant genes among microbiome samples. Here, we summarize existing bioinformatics tools of metagenomic and metatranscriptomic data analysis, the purpose of which is to assist researchers in deciding the appropriate tools for their microbiome studies. Additionally, we propose an Integrated Meta-Function mapping pipeline to incorporate various reference databases and accelerate functional gene mapping procedures for both metagenomic and metatranscriptomic analyses.

Detection techniques of carboxylesterase activity: An update review.

Mammalian carboxylesterases (CESs) are essential members of serine esterase hydrolase superfamily, which are widely distributed in many tissues including liver, intestine, lung and kidney. CESs play an important role in the metabolism of various xenobiotics including ester drugs and environmental toxicants, and also participate in lipid homeostasis, so the development of CESs activity detection techniques are of great significance for drug discovery and biomedical research. With the rapid development of separated and detection technologies such as chromatography, capillary electrophoresis, fluorescent probe-based detection technology, bioluminescent sensor and colorimetric sensor in recent decade, the research of physiological functions of CESs have make huge breakthrough. This review summarizes the development and application of CESs activity detection techniques, as well as comparatively analyzes the characteristics of various detection techniques. The information and knowledge represented here will help the researchers carry out various biochemical studies for understanding activation mechanism and role of CESs in drug metabolism.

Genome-Wide Identification of QTLs for Grain Protein Content Based on Genotyping-by-Resequencing and Verification of qGPC1-1 in Rice.

To clarify the genetic mechanism underlying grain protein content (GPC) and to improve rice grain qualities, the mapping and cloning of quantitative trait loci (QTLs) controlling the natural variation of GPC are very important. Based on genotyping-by-resequencing, a total of 14 QTLs were detected with the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population in 2016 and 2017. Seven of the fourteen QTLs were repeatedly identified across two years. Using three residual heterozygote-derived populations, a stably inherited QTL named as qGPC1-1 was validated and delimited to a ~862 kb marker interval JD1006-JD1075 on the short arm of chromosome 1. Comparing the GPC values of the RIL population determined by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods, high correlation coefficients (0.966 and 0.983) were observed in 2016 and 2017. Furthermore, 12 of the 14 QTLs were identically identified with the GPC measured by the two methods. These results indicated that instead of the traditional KND method, the rapid and easy-to-operate NIRS was suitable for analyzing a massive number of samples in mapping and cloning QTLs for GPC. Using the gel-based low-density map consisted of 208 simple sequence repeat (SSR) and insert/deletion (InDel) markers, the same number of QTLs (fourteen) were identified in the same HHZ/JZ1560 RIL population, and three QTLs were repeatedly detected across two years. More stably expressed QTLs were identified based on the genome resequencing, which might be attributed to the high-density map, increasing the detection power of minor QTLs. Our results are helpful in dissecting the genetic basis of GPC and improving rice grain qualities through molecular assisted selection.

KChIP3 N-Terminal 31-50 Fragment Mediates Its Association with TRPV1 and Alleviates Inflammatory Hyperalgesia in Rats.

Potassium voltage-gated channel interacting protein 3 (KChIP3), also termed downstream regulatory element antagonist modulator (DREAM) and calsenilin, is a multifunctional protein belonging to the neuronal calcium sensor (NCS) family. Recent studies revealed the expression of KChIP3 in dorsal root ganglion (DRG) neurons, suggesting the potential role of KChIP3 in peripheral sensory processing. Herein, we show that KChIP3 colocalizes with transient receptor potential ion channel V1 (TRPV1), a critical molecule involved in peripheral sensitization during inflammatory pain. Furthermore, the N-terminal 31-50 fragment of KChIP3 is capable of binding both the intracellular N and C termini of TRPV1, which substantially decreases the surface localization of TRPV1 and the subsequent Ca2+ influx through the channel. Importantly, intrathecal administration of the transmembrane peptide transactivator of transcription (TAT)-31-50 remarkably reduces Ca2+ influx via TRPV1 in DRG neurons and alleviates thermal hyperalgesia and gait alterations in a complete Freund's adjuvant-induced inflammatory pain model in male rats. Moreover, intraplantar injection of TAT-31-50 attenuated the capsaicin-evoked spontaneous pain behavior and thermal hyperalgesia, which further strengthened the regulatory role of TAT-31-50 on TRPV1 channel. In addition, TAT-31-50 could also alleviate inflammatory thermal hyperalgesia in kcnip3-/- rats generated in our study, suggesting that the analgesic effect mediated by TAT-31-50 is independent of endogenous KChIP3. Our study reveals a novel peripheral mechanism for the analgesic function of KChIP3 and provides a potential analgesic agent, TAT-31-50, for the treatment of inflammatory pain.SIGNIFICANCE STATEMENT Inflammatory pain arising from inflamed or injured tissues significantly compromises the quality of life in patients. This study aims to elucidate the role of peripheral potassium channel interacting protein 3 (KChIP3) in inflammatory pain. Direct interaction of the KChIP3 N-terminal 31-50 fragment with transient receptor potential ion channel V1 (TRPV1) was demonstrated. The KChIP3-TRPV1 interaction reduces the surface localization of TRPV1 and thus alleviates heat hyperalgesia and gait alterations induced by peripheral inflammation. Furthermore, the transmembrane transactivator of transcription (TAT)-31-50 peptide showed analgesic effects on inflammatory hyperalgesia independently of endogenous KChIP3. This work reveals a novel mechanism of peripheral KChIP3 in inflammatory hyperalgesia that is distinct from its classical role as a transcriptional repressor in pain modulation.

Design, synthesis and biological evaluation of novel indole derivatives as potential HDAC/BRD4 dual inhibitors and anti-leukemia agents.

HDAC inhibitors and BRD4 inhibitors were considered to be potent anti-cancer agents. Recent studies have demonstrated that HDAC and BRD4 participate in the regulation of some signal paths like PI3K-AKT. In this work, a series of indole derivatives that combine the inhibitory activities of BRD4 and HDAC into one molecule were designed and synthesized through the structure-based design method. Most compounds showed potent HDAC inhibitory activity and moderate BRD4 inhibitory activity. In vitro anti-proliferation activities of the synthesized compounds were also evaluated. Among them, 19f was the most potent inhibitor against HDAC3 with IC50 value of 5 nM and BRD4 inhibition rate of 88% at 10 µM. It was confirmed that 19f could up-regulate the expression of Ac-H3 and reduce the expression of c-Myc by western blot analysis. These results indicated that 19f was a potent dual HDAC/BRD4 inhibitor and deserved further investigation.

A Case of Alveolar Echinococcosis Occurring in the Hilar Bile Duct.

Echinococcosis is a disease caused by the Echinococcus species that parasitizes in humans. Alveolar echinococcosis (AE) which is caused by Echinococcus multilocularis is harmful to humans. AE mainly occurs in the liver and can be transferred to retroperitoneal lymph nodes, lung, brain, bone, spleen and other organs through lymphatic and blood vessels. Cholangiocarcinoma can occur in the intrahepatic and extrahepatic bile ducts and is more common in the hilar. We reported a case of hilar bile duct alveolar echinococcosis which was originally misdiagnosed an cholangiocarcinoma.

[Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology].

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.