N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71.

Chemical modifications are important for RNA function and metabolism. N4-acetylcytidine (ac4C) is critical for the translation and stability of mRNA. Although ac4C is found in RNA viruses, the detailed mechanisms through which ac4C affects viral replication are unclear. Here, we reported that the 5' untranslated region of the enterovirus 71 (EV71) genome was ac4C modified by the host acetyltransferase NAT10. Inhibition of NAT10 and mutation of the ac4C sites within the internal ribosomal entry site (IRES) suppressed EV71 replication. ac4C enhanced viral RNA translation via selective recruitment of PCBP2 to the IRES and boosted RNA stability. Additionally, ac4C increased the binding of RNA-dependent RNA polymerase (3D) to viral RNA. Notably, ac4C-deficient mutant EV71 showed reduced pathogenicity in vivo. Our findings highlighted the essential role of ac4C in EV71 infection and provided insights into potential antiviral treatments.

An induced-fit de novo initiation mechanism suggested by a pestivirus RNA-dependent RNA polymerase.

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a 'priming element' (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665-680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar 'induced-fit' mechanisms may be commonly taken by PE-containing de novo viral RdRPs.

OsFLA1 encodes a fasciclin-like arabinogalactan protein and affects pollen exine development in rice.

KEY MESSAGE: OsFLA1 positively regulates pollen exine development, and locates in the cellular membrane. Arabinogalactan proteins are a type of hydroxyproline-rich glycoprotein that are present in all plant tissues and cells and play important roles in plant growth and development. Little information is available on the participation of fasciclin-like arabinogalactan proteins in sexual reproduction in rice. In this study, a rice male-sterile mutant, osfla1, was isolated from an ethylmethanesulfonate-induced mutant library. The osfla1 mutant produced withered, shrunken, and abortive pollen. The gene OsFLA1 encoded a FLA protein and was expressed strongly in the anthers in rice. Subcellular localization showed that OsFLA1 was located in the cellular membrane. In the osfla1 mutant, abnormal Ubisch bodies and a discontinuous nexine layer of the microspore wall were observed, which resulted in pollen abortion and ultimately in male sterility. The results show the important role that OsFLA1 plays in male reproductive development in rice.

A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production.

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

Percutaneous Interstitial Nd:YAG Laser Therapy for Axillary Osmidrosis.

BACKGROUND AND OBJECTIVES: To investigate the effects of percutaneous interstitial Nd:YAG laser irradiation on the apocrine glands and molecules involved in odor production (apolipoprotein [ApoD], androgen receptor [AR]) in the subcutaneous tissue of a pig. STUDY DESIGN/MATERIALS AND METHODS: Skin on the back of healthy adult miniature pigs was exposed to pulsed Nd:YAG laser irradiation at 5 or 10 W, or continuous Nd:YAG laser irradiation at 10 W. Samples were taken 1 hour, 1 week, and 1 month after treatment for histology, western blot, and real-time quantitative polymerase chain reaction (qRT-PCR) analysis. RESULTS: One week and 1 month after irradiation, the apocrine glands in pigskin became rounded, glandular cells were shorter, and the glandular cavities were larger compared with controls, but there were no obvious changes in fat cell distribution of collagen around the apocrine glands. One month after irradiation at 10 W in continuous mode, there was a significant decrease in ApoD expression in apocrine cells and ApoD and AR protein and expression levels in pigskin compared with controls. There were also significant differences in ApoD and AR protein and expression levels between treatments. CONCLUSIONS: Percutaneous interstitial Nd:YAG laser irradiation has potential as a safe and efficacious treatment for axillary osmidrosis as it may decrease the production of volatile unsaturated fatty acids, steroids, and associated unpleasant odors in the axilla. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

A unique intra-molecular fidelity-modulating mechanism identified in a viral RNA-dependent RNA polymerase.

Typically not assisted by proofreading, the RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses may need to independently control its fidelity to fulfill virus viability and fitness. However, the precise mechanism by which the RdRP maintains its optimal fidelity level remains largely elusive. By solving 2.1-2.5 Å resolution crystal structures of the classical swine fever virus (CSFV) NS5B, an RdRP with a unique naturally fused N-terminal domain (NTD), we identified high-resolution intra-molecular interactions between the NTD and the RdRP palm domain. In order to dissect possible regulatory functions of NTD, we designed mutations at residues Y471 and E472 to perturb key interactions at the NTD-RdRP interface. When crystallized, some of these NS5B interface mutants maintained the interface, while the others adopted an 'open' conformation that no longer retained the intra-molecular interactions. Data from multiple in vitro RdRP assays indicated that the perturbation of the NTD-RdRP interactions clearly reduced the fidelity level of the RNA synthesis, while the processivity of the NS5B elongation complex was not affected. Collectively, our work demonstrates an explicit and unique mode of polymerase fidelity modulation and provides a vivid example of co-evolution in multi-domain enzymes.

Copper Nanowires Modified with Graphene Oxide Nanosheets for Simultaneous Voltammetric Determination of Ascorbic Acid, Dopamine and Acetaminophen.

Copper nanowires (Cu NWs) were modified with graphene oxide (GO) nanosheets to obtain a sensor for simultaneous voltammetric determination of ascorbic acid (AA), dopamine (DA) and acetaminophen (AC). The nanocomposite was obtained via sonication, and its structures were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS). The electrochemical oxidation activity of the materials (placed on a glassy carbon electrode) was studied by cyclic voltammetry and differential pulse voltammetry. Due to the synergistic effect of Cu NWs and GO, the specific surface, electrochemical oxidation performance and conductivity are improved when compared to each individual component. The peaks for AA (-0.08 V), DA (+0.16 V), and AC (+0.38 V) are well separated. The sensor has wide linear ranges which are from 1-60 µM, 1-100 µM, and 1-100 µM for AA, DA, and AC, respectively, when operated in the differential pulse voltammetric mode. The detection limits are 50, 410 and 40 nM, respectively. Potential interferences by uric acid (20 µM), glucose (10 mM), NaCl (1 mM), and KCl (1 mM) were tested for AA (1 µΜ), DA (1 µΜ), and AC (1 µΜ) and were found to be insignificant. The method was successfully applied to the quantification of AA, DA, and AC in spiked serum samples.

Prognostic value of somatosensory evoked potentials, neuron-specific enolase, and S100 for short-term outcome in ischemic stroke.

To predict short-term outcome in acute ischemic stroke, we analyzed somatosensory evoked potentials (SEP) and biochemical parameters [neuron-specific enolase (NSE) and S100 protein] in a prospective study with serial measurement. In 31 patients with 1st middle cerebral artery infarction, serum NSE and S100 protein were measured daily between days 1 and 6 poststroke. The N20 and N70 components of the SEP (SEP20 and SEP70) were determined on days 1 and 6. SEP and biochemical markers in stroke patients were compared with a control group. Short-term outcome was assessed by the modified Rankin Scale (mRS) at days 7-10 and was dichotomized between good (mRS 0-2) and poor (mRS ≥3) outcome. Specificity and positive predictive value (PPV) were high at day 1 for SEP (SEP20: 100% for both; SEP70: 93 and 88%, respectively) compared with lower values for NSE (67 and 50%) and S100 (23 and 57%). In contrast, S100 showed the highest sensitivity at day 1 with 77% compared with a relatively low sensitivity of NSE (31%) and SEP (SEP20: 35%, SEP70: 47%). The biochemical markers showed an improving sensitivity over time with best values (>90%) between days 3 and 4 at the expense of a lower specificity. Specificity and PPV of SEP on day 6 was still 100% with sensitivity increasing up to 53% (SEP20) and 60% (SEP70). SEP could early differentiate between good and poor outcome and reliably predict poor outcome. Since biochemical markers and SEP complement each other in the prognosis of stroke, a combined application of these markers seems promising.

A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family.

RNA-dependent RNA polymerases (RdRPs) from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A-E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

Prognostic value of electroencephalography and evoked potentials in the early course of malignant middle cerebral artery infarction.

Space-occupying brain edema may lead to a malignant course in patients with large middle cerebral artery infarction. Decompressive hemicraniectomy has to be initiated early to prevent further tissue damage. In this retrospective study, we analyzed electroencephalography (EEG) and evoked potentials (EPs), obtained within 24 h after onset of stroke, in 22 patients suffering from a large middle cerebral artery infarction. Our findings indicate a prognostic value of EEG and brainstem auditory EP (BAEP): the absence of delta activity and the presence of theta and fast beta frequencies within EEG-focus predicted a non-malignant course. In contrast, diffuse generalized slowing and slow delta activity in the ischemic hemisphere pointed to a malignant course. Likewise, pathological BAEP were correlated with a malignant course. The coexistence of background slowing and pathological BAEP showed the highest level of significance. In conclusion, our findings implicate an additional early application of electrophysiological methods in stroke patients. EEG and EP deliver useful information to select those patients who develop malignant edema.

OsCCRL1 is Essential for Phenylpropanoid Metabolism in Rice Anthers.

Phenylpropanoid metabolism and timely tapetal degradation are essential for anther and pollen development, but the underlying mechanisms are unclear. In the current study, to investigate this, we identified and analyzed the male-sterile mutant, osccrl1 (cinnamoyl coA reductase-like 1), which exhibited delayed tapetal programmed cell death (PCD) and defective mature pollen. Map-based cloning, genetic complementation, and gene knockout revealed that OsCCRL1 corresponds to the gene LOC_Os09g32020.2, a member of SDR (short-chain dehydrogenase/reductase) family enzyme. OsCCRL1 was preferentially expressed in the tapetal cells and microspores, and localized to the nucleus and cytoplasm in both rice protoplasts and Nicotiana benthamiana leaves. The osccrl1 mutant exhibited reduced CCRs enzyme activity, less lignin accumulation, delayed tapetum degradation, and disrupted phenylpropanoid metabolism. Furthermore, an R2R3 MYB transcription factor OsMYB103/OsMYB80/OsMS188/BM1, involved in tapetum and pollen development, regulates the expression of OsCCRL1. Finally, the osmyb103 osccrl1 double mutants, exhibited the same phenotype as the osmyb103 single mutant, further indicating that OsMYB103/OsMYB80/OsMS188/BM1 functions upstream of OsCCRL1. These findings help to clarify the role of phenylpropanoid metabolism in male sterility and the regulatory network underlying the tapetum degradation.

[Indwelling the anterior urethral stent: a good treatment option for anterior urethral stricture].

OBJECTIVE: To observe the clinical effect of indwelling the anterior urethral stent in the treatment of anterior urethral stricture. METHODS: We included 38 patients with anterior urethral stricture in the treatment group, and another 38 in the control, the former treated by indwelling the anterior urethral stent, and the latter by urethral dilatation. Then we analyzed the clinical results by comparing the Qmax, urinary hesitancy and numbers of urethral dilations between the two groups. RESULTS: Compared with the controls, the patients of the treatment group showed an obvious increase in Qmax, a significant decrease in the number of urethral dilatations, and a marked improvement of the quality of life. Six months after the stent removal, there were significantly more patients with Qmax > 15 ml/s in the treatment group than in the control (P < 0.05). CONCLUSION: Indwelling the anterior urethral stent is a desirable option for the treatment of anterior urethral stricture, which is simple, safe, effective and reliable, and can be applied to general clinical practice.

Botulinum toxin A for neuropathic pain after neck dissection: a dose-finding study.

OBJECTIVES: Botulinum toxin type A (BtxA) has been reported to be feasible in chronic neuropathic pain after neck dissection. The impact of the dose on the outcome has not been investigated yet. STUDY DESIGN: Twenty-three patients with neuropathic pain after neck dissection were selected for an open and prospective phase II trial. METHODS: In the low-dose group (n=13), a concentration of 10 mouse units (MU)/0.1 mL saline and in the high-dose-group (n=10), a concentration of 20 MU/0.1 mL saline were injected subcutaneously. Pain and quality of life were assessed at day 0 and day 28, respectively, by visual analog scales (VAS) and European Organization for Research and Treatment of Cancer (EORTC) quality-of-life core and EORTC quality-of-life head and neck module questionnaires. RESULTS: Patients in the low-dose group showed a significant pain reduction (VAS) from 4.3 at day 0 to 3.0 at day 28 (P<.05). The mean pain VAS values in the high-dose group did not improve significantly. No serious adverse events were observed. There were trends toward improvement in quality of life in the low-dose group. CONCLUSIONS: BtxA in a low concentration seems to be a useful therapeutic option in chronic neuropathic pain of the neck and shoulder after neck dissection.

Outpatient procedures on the glans and anterior urethra under a new local anaesthesia: intracorpus spongiosum anaesthesia.

OBJECTIVE: To determine the efficacy of intracorpus spongiosum anaesthesia during minor procedures on the glans and anterior urethra in the outpatient clinic. METHODS: Sixty-nine consecutive male patients underwent various procedures on the glans penis or anterior urethra under intracorpus spongiosum anaesthesia, which was performed by injection of 3 mL of 1% lidocaine into the glans penis. The effect of this anaesthetic technique was assessed by questionnaire using a pain scale. RESULTS: Following injection of lidocaine, the anaesthetic effect was immediate and very satisfactory. The minor procedures, varying from 8 to 68 minutes in duration, were successfully completed under the new local anaesthesia. Of the 69 patients, 63 (91.3%) felt no pain and six (8.7%) reported either minor or moderate discomfort that was tolerable and could be ignored. There were no serious complications with this anaesthetic technique except for three patients (4.3%) who had instantaneous trance during lidocaine injection. CONCLUSION: Intracorpus spongiosum anaesthesia is an effective, simple, safe anaesthetic technique for minor procedures on the glans and anterior urethra in an outpatient setting. With this new local anaesthesia, the total cost of many procedures on the glans and anterior urethra can be markedly reduced.

[In vivo HSV-TK gene transfer and the therapeutic effects of ganciclovir on bladder cancer in mice with HSV-TK gene transfer].

OBJECTIVE: To explore an effective method for in vivo HSV-TK gene transfer and observe the therapeutic effect of ganciclovir (GCV) on bladder cancer in mice after receiving such gene transfer therapy. METHODS: Mice models bearing subcutaneous tumors of bladder cancer were established in 24 syngeneic mice, and HSV-TK gene transfer was performed by means of naked DNA plasmids, DNA-liposome complex or retroviral mediators respectively. GCV treatment was administered in all the mice and its effect on the growth of the tumor was subsequently observed. RESULTS: Gene transfer therapies by DNA-liposome complex and retroviral mediators both significantly inhibited the growth of the tumor by 54% and 68% respectively, and the survival of the mice was prolonged by 28.81% and 44.16% (P<0.05). The effect of gene transfer by naked DNA plasmids was not obvious (P>0.05). CONCLUSION: Gene transfer therapies by DNA-liposome complex and retroviral vectors supplemented by GCV treatment are effective therapeutic modalities against bladder cancer.

Evoked potentials in acute ischemic stroke within the first 24 h: possible predictor of a malignant course.

OBJECTIVE: In patients with large middle cerebral artery (MCA) infarction, space-occupying brain edema may lead to a malignant course with up to 80% mortality, under conservative treatment. As interventional treatment strategies must be started before the deterioration occurs, predictors of a malignant course are necessary. MATERIAL AND METHODS: This study reports on the results of evoked potentials (brainstem auditory evoked potentials [BAEP] and median-nerve somatosensory evoked potentials [SEP]) within 24 h after onset of stroke in 30 patients suffering a large MCA infarct (14 patients with a malignant and 16 with a non-malignant course). RESULTS: Our findings indicate that pathological BAEP indicate a malignant course (P < 0.05). CONCLUSION: This study shows that in patients suffering from large MCA infarction early assessment of EP within 24 h after onset of stroke may deliver useful information to select those patients who develop malignant edema.

Belly dancer's syndrome following central pontine and extrapontine myelinolysis.

We report on a woman with delayed-onset of belly dancer's syndrome 5 months after central pontine and extrapontine myelinolysis (CPM/EPM) and severe hyponatriemia. This case demonstrates that basal ganglia lesions in EPM can be the underlying pathoanatomic substrate for the rarely observed belly dancer's syndrome. The sequential appearance of extrapyramidal symptoms might reflect an ongoing but ineffective or deficient remyelination process. The presence of CPM/EPM should be considered in patients with involuntary dyskinesias of the abdominal wall.

Botulinum toxin type A for the treatment of chronic neck pain after neck dissection.

BACKGROUND: Neck dissection surgery is often followed by chronic head and neck pain. To date optimal treatment of this type of pain is lacking. Botulinum toxin type A (BTX-A) has been shown to be effective in the treatment of myofascial pain syndrome and headache. In a pilot study, we wanted to test the effectiveness of BTX-A for the treatment of chronic neck pain after neck dissection. METHODS: Sixteen patients with chronic neck pain after neck dissection were included in this prospective, open study. Eighty to 320 units of BTX-A (Dysport) were injected into muscular trigger points. Outcome measures included chronic pain and shooting pain on the basis of visual analog scales and quality of life improvement (EORTC QLQ-C-30; EORTC QLQ-H and N35) before and 4 weeks after treatment. RESULTS: Patients showed a significant reduction in chronic pain (4.5 before to 3.3 after treatment, p =,005) and in shooting pain (6.1 before to 4.7 after treatment, p =.005). There was a trend toward improvement in global quality of life (QLQ-C30, p =.097) and an increase on the functional scale "pain" (QLQ-H and N35, p =.071). CONCLUSIONS: BTX-A treatment of subjects with chronic neck pain after neck dissection resulted in a fast and significant reduction of pain. A significant improvement in quality of life may be expected in a longer time course after treatment.