MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most upregulated miRs in tubers. Here, we characterised miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a. METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared with autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH-SY5Y cells following miR-34a transfection. RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR-34a was also strongly expressed in foetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. CONCLUSIONS: MicroRNA-34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR-34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC.

Production and characterization of active recombinant human factor II with consistent sialylation.

Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has 10 sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation. In order to develop such a fed-batch process, various process parameters were evaluated to determine their impact on product quality. Process temperature and temperature shift timing were important for both sialic acid level and gamma-carboxyglutamate (Gla) level. In addition, vitamin K concentration and the type of surfactant used for preparation of vitamin K stock solution were also important for gamma carboxylation. A fed-batch study performed with various medium additives known to be involved in the N-glycosylation pathway, such as N-acetyl-d-mannosamine (ManNAc), galactose (Gal), dexamethasone, and manganese sulfate, increased the level of sialylation and enabled the elucidation of some potential bottlenecks in the sialylation pathway. The optimized process based on these studies yielded a reduction in the level of missing Gla by 0.4 moles per mole of rhFII in cell culture and a nearly threefold increase in sialic acid level. The process was successfully implemented at the 2000 L scale where a high Gla level and sialylation levels were achieved in all GMP lots. Biotechnol. Bioeng. 2017;114: 1991-2000. © 2017 Wiley Periodicals, Inc.

Preoperative Three-Dimensional Simulation of Osteotomy for Correction of Malunion of Both Bones of Forearm: A Case Report.

Corrective osteotomy of malunion of both forearm bones is a challenging procedure because it needs accurate angular and rotational correction of both bones. Recent advances in three-dimensional (3D) printing techniques have shown promising results in the correction of complex deformities. We report a patient with malunion of both bones of the forearm in whom we determined site and degree of correction required based on the computed tomography images of the affected side and mirrored images of the contralateral healthy side. We then created 3D printed sawbones and simulated osteotomy to confirm stable dynamic forearm rotation. This method enabled satisfactory restoration of anatomical and functional outcomes. Preoperative dynamic motion simulation using 3D printed anatomic bone model is helpful for complex corrective osteotomy of forearm fracture malunion. Level of Evidence: Level V (Therapeutic).

Arc Centre Distance Change after Treatment of Complex Transverse Olecranon Fractures with a Large Impacted Articular Fragment Treated with Tension Band Wiring and Bone Graft.

Background: The treatment of complex transverse olecranon fractures (CTOFs), also known as olecranon fractures with the impacted articular fragments (IAF), was reported recently. We fix IAFs with absorbable pins supported by autologous bone graft followed by olecranon fracture fixation. We have used the arc centre distance (ACD) method to evaluate the congruency of this concentric hinge joint. The aim of this study is to present the outcomes of this fixation technique and evaluate the value of the ACD method. Methods: We reviewed 26 cases of CTOF treated at our hospital from 2014 to 2020. The functional outcome and range of motion of the elbow joint was measured by MEPS (Mayo Elbow Performance Score). We measured the ACD of each fragment of the ulnotrochlear joint [coronoid process (CP), IAF and olecranon process (OP)] with the CT image taken at 15 months postoperatively on average. Results: The mean arc of elbow motion was 3º to 132º. The mean MEPS at 1 year postoperatively was 94, and 25 of 26 cases (96%) achieved a good or excellent outcome. Twelve patients, who took the elbow CT at least more than 3 months post-operatively, were included for ACD measurement. The postoperative joint incongruency of each fragment was as follows, 0.39 ± 0.70, 0.40 ± 0.69 and 0.29 ± 0.72 mm (CP, IAF and OP, respectively) according to the ACD method. The ACD value for each fragment was significantly different before and after the surgery (p < 0.05). Conclusions: Accurate reduction of IAF with absorbable pins completed by tension band wiring with autogenous bone grafting can be an effective technique for CTOF with a large IAF. The restoration of the joint improved ACD. Level of Evidence: Level IV (Therapeutic).

Cooperativity of Kv7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells.

The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered Kv7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-Kv7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered Kv7.4 channels mechanically sensitive, confirmed in consolidated Kv7.4 channels at the base of OHCs. Kv7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast Kv7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies.

The anterior approach for conversion hip arthroplasty.

Conversion of prior proximal femoral fracture fixation to hip arthroplasty is a fairly common and successful procedure, necessitated by various modes of failure. The procedure is well described utilizing a posterior or anterolateral surgical approach. The anterior approach for total hip arthroplasty has gained in popularity. The approach allows for supine positioning and facilitates live fluoroscopic imaging. We present possible advantages and disadvantages, as well as the surgical technique, of conversion to total hip arthroplasty via the direct anterior approach.

Implementation of Fully Integrated Continuous Antibody Processing: Effects on Productivity and COGm.

The changing landscape of the biopharmaceutical market is driving a paradigm shift toward continuous manufacturing. To date, integrated continuous bioprocessing has not been realized as enabling technologies are nascent. In this work, a fully integrated continuous process is successfully demonstrated from pilot scale bioreactor to drug substance. Comparable product quality is observed between the continuous process and a 500 L fed-batch conventional process. The continuous process generated material at a rate of 1 kg of purified mAb every 4 days, achieving a 4.6-fold increase in productivity compared to the fed-batch process A plant throughput analysis using BioSolve software shows that a fed-batch facility with 4 × 12 500 L stainless steel bioreactors and purification train of the corresponding scale can be replaced by a continuous facility consisting of 5 × 2000 L single use bioreactors and smaller purification train, with a cost reduction of 15%.

Single-Cell RNA-seq Reveals Profound Alterations in Mechanosensitive Dorsal Root Ganglion Neurons with Vitamin E Deficiency.

Ninety percent of Americans consume less than the estimated average requirements of dietary vitamin E (vitE). Severe vitE deficiency due to genetic mutations in the tocopherol transfer protein (TTPA) in humans results in ataxia with vitE deficiency (AVED), with proprioceptive deficits and somatosensory degeneration arising from dorsal root ganglia neurons (DRGNs). Single-cell RNA-sequencing of DRGNs was performed in Ttpa-/- mice, an established model of AVED. In stark contrast to expected changes in proprioceptive neurons, Ttpa-/- DRGNs showed marked upregulation of voltage-gated Ca2+ and K+ channels in mechanosensitive, tyrosine-hydroxylase positive (TH+) DRGNs. The ensuing significant conductance changes resulted in reduced excitability in mechanosensitive Ttpa-/- DRGNs. A highly supplemented vitE diet (600 mg dl-α-tocopheryl acetate/kg diet) prevented the cellular and molecular alterations and improved mechanosensation. VitE deficiency profoundly alters the molecular signature and functional properties of mechanosensitive TH+ DRGN, representing an intriguing shift of the prevailing paradigm from proprioception to mechanical sensation.

Autocatalytic activation of influenza hemagglutinin.

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.

Collagenous micronodules in prostate cancer revisited: are they solely associated with Gleason pattern 3 adenocarcinomas?

Collagenous micronodules (CMs) are microscopic stromal nodular eosinophilic fibrillar collagen deposition of uncertain histogenesis seen in prostatic adenocarcinoma. Per the 2005 International Society of Urologic Pathology (ISUP) consensus conference, they are categorized as Gleason pattern 3. This study analyzes morphological and clinical features of CMs from a large series of radical prostatectomies. Hematoxylin and eosin stained slides for 129 radical prostatectomies for adenocarcinoma of prostate with CMs and for 93 prostatic adenocarcinoma cases without CMs as comparison were examined out of a total of 667 cases performed from January 2010 to December 2011 at Houston Methodist Hospital. CMs were identified in 19% of all radical prostatectomies (129/667 cases). Almost all tumors with CMs were located in the peripheral zone (98%) as single or multiple foci of prostatic cancer glands. The vast majority of cases (96%) were identified in association with mucinous secretion. A cribriform Gleason pattern 4 was associated in 86 cases (67%). The CMs were associated with glomerulation (42%) and amphophilic luminal secretion (59%). 88 cases (68%) showed tumor foci with Gleason pattern ≥ 4 in close association with CMs. Multivariate analysis revealed CMs of the prostatic adenocarcinoma are closely related to mucinous secretion, cribriform growth pattern, and Gleason pattern 4. This study suggests that CMs are more frequently associated with Gleason pattern 4 cancer warranting morphologic reappraisal of CMs, rather than the consensus assignment of Gleason pattern 3.

The clinical significance of findings obtained on 3D-FLAIR MR imaging in patients with Ramsay-Hunt syndrome.

OBJECTIVES/HYPOTHESIS: To investigate the clinical significance of three-dimensional-fluid-attenuated inversion recovery (3D-FLAIR) magnetic resonance imaging (MRI) findings in patients with Ramsay-Hunt syndrome (RHS). STUDY DESIGN: Case series. METHODS: We enrolled 28 consecutive patients of RHS with temporal bone MRI. Initial clinical findings and outcome were assessed by House-Brackmann (HB) scales, electroneuronography (ENoG), and pure tone audiometry (PTA). Two radiologists evaluated the presence of abnormalities on pre-/postcontrast 3D-FLAIR for the cranial nerve (CN)-VII, CN-VIII, inner ear (IE), and the posterior fossa by consensus. The relative signal intensity and enhancement degree (rED) of the structures were measured using ImageJ (http://rsbweb.nih.gov/ij/). Statistical test correlated the clinical symptoms and the outcome with the analysis results of 3D-FLAIR images. RESULTS: 3D-FLAIR demonstrated enhancement of CN-VII in all patients. Precontrast hyperintensity and enhancement were seen in eight and 16 patients with IE, and in four and six with CN-VIII, respectively. Precontrast hyperintensity of IE or CN-VIII was significantly associated with the presence of vertigo (P value < 0.05). Precontrast hyperintensity of IE or CN-VIII significantly correlated with clinical symptoms assessed by HB, ENoG, and PTA (P value < 0.05, respectively). rED of the vestibule moderately correlated with initial HB scale (r = 0.391, P = 0.039). There was no correlation between any of the 3D-FLAIR findings and the follow-up HB. CONCLUSIONS: RHS shows frequent abnormalities of IE or CN-VIII, as well as CN-VII on pre-/postcontrast 3D-FLAIR images. Precontrast hyperintensity of IE/CN-VIII on 3D-FLAIR is significantly correlated with the severity of facial palsy, the presence of vertigo, and the degree of hearing impairment but not with clinical outcome.

Genomic analysis of stress response against arsenic in Caenorhabditis elegans.

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.

Genomic analysis of immune response against Vibrio cholerae hemolysin in Caenorhabditis elegans.

Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as pore-forming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes.Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans.

The dose effect of ephedrine on the onset time of vecuronium.

UNLABELLED: A small dose of ephedrine decreases the onset time of rocuronium and cisatracurium; however, ephedrine might be associated with adverse hemodynamic effects. The appropriate dose of ephedrine has not been determined. We, therefore, studied 120 patients anesthetized with fentanyl 2 microg/kg and propofol 2-2.5 mg/kg who were randomly divided to receive either ephedrine (30, 70, or 110 microg/kg) or saline. During propofol anesthesia, the neuromuscular block was monitored by mechanomyography by using submaximal current of train-of-four stimulation every 10 s. To determine cardiac output, a transcutaneous Doppler probe was placed externally at the suprasternal notch. Tracheal intubation was performed by a blinded investigator at 2 min after vecuronium. Neuromuscular block, intubating conditions, and hemodynamic effects were measured during the induction of anesthesia. Both ephedrine 70 and 110 microg/kg improved intubating conditions at 2 min after vecuronium; however, 110 microg/kg was associated with adverse hemodynamic effects. We conclude that ephedrine 70 microg/kg given before the induction of anesthesia improved intubating conditions at 2 min after vecuronium, probably by increased cardiac output without significant adverse hemodynamic effects. IMPLICATIONS: Ephedrine 70 microg/kg given before the induction of anesthesia improved tracheal intubating conditions at 2 min after vecuronium by increased cardiac output without significant adverse hemodynamic effects.