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Pregnancy is a complicated process involving various anatomical and physiological changes to provide a suitable environment for fetal development, to meet the increased metabolic demands, and to prepare for labor. While most pregnancies and births are uneventful, all pregnancies are at risk of complications. No less than 15% of all pregnant women develop a potentially lifethreatening complication or may also experience an unpleasant outcome. Conventional sequencing has been widely used in this field for diagnoses and treatment planning. However, it lacks the precise resolution to identify transcriptomic variation between the nearby cell types. Single-cell RNA sequencing is a novel approach that through single-cell isolation, transcript capturing, and development and sequencing of expression libraries allows the assessments of fundamental biological properties of cell populations and biological systems at unprecedented resolution. The single-cell sequencing technology is a powerful tool for investigating cell distribution and cell-to-cell relationships in human reproduction. Since 2016, the technique has contributed to the discovery of many transcriptomic atlases of human embryos, placenta, decidua, and endometrium and the identification of various crucial regulatory pathways in fetal development. Here, we briefly describe the application of single-cell RNA sequencing in the studies of physiological processes and pathological mechanisms of pregnancy.

Problem Various etiological factors, such as infection and inflammation, may induce the adverse outcomes of pregnancy of miscarriage, stillbirth, or preterm birth. The pathogenic mechanisms associated with these adverse pregnancies are yet unclear. We hypothesized that a common pathogenic mechanism may underlie variant adverse outcomes of pregnancy, which are induced by genetic-environmental factors. The specific objective of the current study is to uncover the common molecular mechanism(s) by identifying the specific transcripts that are present in variant subtypes of pregnancy loss and preterm birth. Method of study Transcriptomic profiling was performed with RNA expression microarray or RNA sequencing of placentas derived from pregnancy loss (which includes spontaneous miscarriage, recurrent miscarriage, and stillbirth) and spontaneous preterm birth, followed by bioinformatic analysis of multi-omic integration to identify pathogenic molecules and pathways involved in pathological pregnancies. Results The enrichment of common differentially expressed genes between full-term birth and preterm birth and pregnancy loss of miscarriage and stillbirth revealed different pathophysiological pathway(s), including cytokine signaling dysregulated in spontaneous preterm birth, defense response, graft-versus-host disease, antigen processing and presentation, and T help cell differentiation in spontaneous miscarriage. Thirty-three genes shared between spontaneous preterm birth and spontaneous miscarriage were engaged in pathways of interferon gamma-mediated signaling and of antigen processing and presentation. For spontaneous miscarriage, immune response was enriched in the fetal tissue of chorionic villi and in the maternal facet of the placental sac. The transcript of nerve growth factor receptor was identified as the common molecule that is differentially expressed in all adverse pregnancies: spontaneous preterm birth, stillbirth, spontaneous miscarriage, and recurrent miscarriage. Superoxide dismutase 2 was up-regulated in all adverse outcomes of pregnancy except for recurrent miscarriage. Cytokine-cytokine receptor interaction was the common pathway in spontaneous preterm birth and spontaneous miscarriage. Defense response was enriched in the fetal tissue of miscarriage and in the maternal tissue in spontaneous miscarriage. Conclusions Our results indicated that the chemokine-cytokine pathway may play important roles in and function as a common pathogenic mechanism associated with, the different adverse outcomes of pregnancy, which demonstrated that differentially expressed transcripts could result from a common pathogenic mechanism associated with pregnancy loss and spontaneous preterm birth, although individual pregnancy outcomes may differ from each other phenotypically.

Background: Congenital heart defects (CHDs) are the most common cause of birth defects. Assessment of the incidence, distribution, disease spectrum, and genetic deficits of fetal CHDs in China is urgently needed. Methods: A national echocardiography screening program for fetal CHDs was implemented in 92 prenatal screening–diagnostic centers in China. Findings: A total of 18,171 fetal CHD cases were identified from 2,452,249 pregnancies, resulting in 7·4/1,000 as the national incidence rate of fetal CHD. The incidences of fetal CHD in the six geographical regions, the southern, central, eastern, southwestern, northern, and northwestern, were 8·174 (CI: 7·802–8·554), 7·839 (CI: 7·680–8·000), 7·647 (CI: 7·383–7·915), 7·562 (CI: 7·225–7·907), 5·618 (CI: 5·337–5·906), and 4·716 (CI: 4·341–5·108), respectively, per 1,000 pregnancies. A total of 76·24% cases of fetal CHD were found to be an isolated intracardiac single defect. Among all extracardiac malformations, the CNS was the most common tissue with extracardiac anomalies associated with CHD, accounting for 22·89% of fetal CHD cases. Chromosomal karyotyping identified trisomy 18 as the most common chromosomal abnormality in fetal CHD. Molecular genetic study of copy number variations characterized microdeletion and/or microrepetition/duplication and uniparental disomy. CHD-associated syndromes could be identified with a comprehensive approach integrating prenatal ultrasound, MRI, pathological autopsy, and cytogenetics and molecular genetics. Interpretation: Implementation of prenatal echocardiography is a practically feasible platform to screen fetal CHD, which may reduce the financial and emotional burden of CHD. Funding: This study was supported in part by the Key Project of Science and Technology Planning Project of Hubei Province (2010CDA038), the Hubei Province Natural Science Foundation (2012FFB01203), the Key Project of Hubei Province Natural Science Foundation (2011CDA012 and 2014CFA062), and the New York State Research Foundation for Mental Hygiene (914-3280, 914-3257). Declaration of Interest: All authors have declared that there are no financial conflicts or conflicts of interest with other individuals or organizations. Ethical Approval: The procedures and protocols implemented were reviewed and approved by the Ethics Committees of Maternal and Child Health Hospital of Hubei Province.

Rationale: Floating ankle is a rare traumatic condition characterized by a combination of tibial and ipsilateral foot fractures, with the ankle remaining intact. It is usually caused by high-energy trauma and also presents with serious soft tissue damage. Its treatment is mainly restricted to external fixation, which results in poor outcomes. We present a patient with a floating ankle who underwent staged treatment and achieved full internal fixation, subsequently returning to normal activity. Patient concerns: A 26 year- old man had an accident with an reel machine and sustained an open fracture on his right lower extremity. Diagnoses: Digital radiograph demonstrated a distal tibial fracture, fibular fracture, and multiple metatarsal fractures, which fulfilled the criteria for a floating ankle. Interventions: Initial ankle-spanning external fixation was performed. After 21 days, the patient underwent open reduction and internal fixation on his first and fifth metatarsals, and K-wire fixation on his fourth metatarsal. The external fixator was replaced by plaster fixation. Seven days later, the patient underwent internal fixation of his leg, open reduction and internal fixation with plating was applied of the fibular fracture, and minimally invasive plate osteosynthesis of the tibial fracture. Outcomes: At 1-year follow-up, bone union was identified by digital radiograph; after 2 years, his ankle function had fully recovered, and he resumed his normal activities. Lessons: In the staged treatment protocol of the floating ankle, temporary external fixation provided traction and immobilization of the skeletal and soft tissues. Secondary internal fixation maintained the reduction and alignment and allowed early exercise, which is critical to the prognosis of a floating ankle.

Background and purpose N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. Experimental approach Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. Key results The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. Conclusions and implications These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. Linked articles This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

A number of meta-analyses have compared clinical outcomes following plate vs. intramedullary fixation for midshaft clavicle fractures (MSCF), but with conflicting results. There is a requirement for updated level-1 evidence to guide clinicians managing MSCF. The aim of the present systematic review and meta-analysis was to compare clinical outcomes following plate vs. intramedullary fixation of MSCF. The PubMed, Scopus, BioMed Central, Cochrane Central Register of Controlled Trials and Google Scholar databases were searched for records added until 1st July 2019. A total of 10 randomised controlled trials (RCTs) were included. Shoulder function was assessed using the Constant-Murley Shoulder Outcome questionnaire and the Disabilities of the Arm, Shoulder and Hand questionnaire (DASH). There was no statistically significant difference in Constant-Murley scores between plate and intramedullary fixation [Mean difference (MD)=0.75; 95% CI: -2.49 to 3.99; P=0.65; I2=85%]. Similarly, there was no statistically significant difference in DASH scores between the two groups (MD=1.55; 95% CI: -1.12 to 4.23; P=0.26; I2=89%). There was no statistically significant difference in complications requiring non-routine surgery between plate and intramedullary fixation [risk ratio (RR)=1.80, 95%CI: 0.80-4.05, P=0.15; I2=0%]. There was an increased risk of complications that did not require non-routine surgery with plate fixation as compared to intramedullary fixation (RR=2.38, 95%CI: 1.22-4.62, P=0.01; I2=70%). Plate fixation was also associated with an increased risk of infection and complications of cosmetic dissatisfaction. The present results indicated no difference in long-term functional outcomes between plate and intramedullary fixation of MSCF. Plate fixation was associated with an increased risk of complications not requiring non-routine surgery. Further high-quality RCTs shall strengthen the evidence on this subject.

Background: We aimed to assess the psychological burden in healthcare professionals during the COVID-19 outbreak, particularly in locked-down Chinese cities, to provide evidence for exploring early preventive interventions. Methods: A cross-sectional study was designed, with an online questionnaire for data collection on the psychological distress, stress, and anxiety subscales of the Depression Anxiety Stress Scale 21, and on social and environmental factors, particularly the influence of city lockdown, among healthcare professionals exposed to COVID-19, February 4–16, 2020. Findings: Data were collected from 636 medical professionals and 1929 college students. Psychological burden includes psychological distress (unpleasant feelings or emotions that impact normal activities of daily living), stress (physical, mental, or emotional factor that causes bodily or mental tension), and anxiety (a feeling of apprehension and fear, characterized by physical symptoms such as palpitations, sweating, and feelings of stress), accounting for 36·48%, 13·36%, and 25·31%, respectively, among medical professionals. Medical staff with COVID-19 exposure were 2·04 times more likely to be anxious than non-exposed staffs (95% CI 1·14–3·63). The psychological distress of medical staff increased directly with the shortage of medical supplies (AOR=6·70; 95% CI 2·37–18·92). City lockdown was a critical factor that caused psychological burden. Interpretation: Healthcare professionals, especially those with exposure to COVID-19 in locked-down cities, were in a poor psychological state during the COVID-19 outbreak. Intervention and prevention programs that aim to address the psychological burden of healthcare professionals facing emerging infectious diseases are urgently needed. Funding Statement: This study was funded by the Shantou Science and Technology Project [2020] (number 6-5). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The research content and process have been approved by the Institutional Review Board of Shantou University Medical College (Project No. SUMC2020-01). All interviewees provided written informed consent.

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Rationale Tension band wiring is the most widely accepted technique for the treatment of patellar fractures but the technique is associated with common complications like wire migration, prominence, and breakage. To reduce these complications, we developed and propose a modified technique that has a superior biomechanical strength and a potential to reduce such postoperative complications. Patient concerns The patient presented with pain and mild swelling in his left knee after he slipped on the floor and fell on his left knee. He has no significant past medical or surgical history. The patient took the tension band wiring as the first choice because of the wide acceptance. But he worried about the complications. Diagnoses X-ray showed a transverse fracture of the left patella with an inferior pole occult fracture. Interventions The patient was operated with a modified technique of the classic tension band wiring for patellar fractures. In our 4-step procedure, double tension cerclage wires were wrapped under the exposed ends of the Kirschner wires (K-wires) and the tendons in figure-of-8 fashion. The aim was to increase the biomechanical strength so that when one of the tension wires fail, the other one can hold the fragments together. Outcomes The patient recovered very well and without any complications. The patient was followed-up for 1 year and the fracture has united very well, with satisfying knee range of motion. Lessons From this case study, we can detect the biomechanical advantages of our technique which can increase the stability of the fracture and that allows early functional exercise and additionally the micromotion at the fracture site has a beneficial effect of fracture union. Based on the perfect outcomes, our technique is worthy of clinical application.

Astrocytes, which are five-fold more numerous than neurons in the central nervous system (CNS), are traditionally viewed to provide simple structural and nutritional supports for neurons and to participate in the composition of the blood brain barrier (BBB). In recent years, the active roles of astrocytes in regulating cerebral blood flow (CBF) and in maintaining the homeostasis of the tripartite synapse have attracted increasing attention. More importantly, astrocytes have been associated with the pathogenesis of Alzheimer’s disease (AD), a major cause of dementia in the elderly. Although microglia-induced inflammation is considered important in the development and progression of AD, inflammation attributable to astrogliosis may also play crucial roles. A1 reactive astrocytes induced by inflammatory stimuli might be harmful by up-regulating several classical complement cascade genes thereby leading to chronic inflammation, while A2 induced by ischemia might be protective by up-regulating several neurotrophic factors. Here we provide a concise review of the emerging roles of astrocytes in the homeostasis maintenance of the neuro-vascular unit (NVU) and the tripartite synapse with emphasis on reactive astrogliosis in the context of AD, so as to pave the way for further research in this area, and to search for potential therapeutic targets of AD.

We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.

Rationale: Anatomical characteristics, such as subcutaneous position and minimal muscle cover, contribute to the complexity of fractures of the distal third of the tibia and fibula. Severe damage to soft tissue and instability ensure high risk of delayed bone union and wound complications such as nonunion, infection, and necrosis. Patient concerns: This case report discusses management in a 54-year-old woman who sustained fractures of the distal third of the left tibia and fibula, with damage to overlying soft tissue (swelling and blisters). Plating is accepted as the first choice for this type of fracture as it ensures accurate reduction and rigid fixation, but it increases the risk of complications. Diagnosis: Closed fracture of the distal third of the left tibia and fibula (AO: 43-A3). Interventions: After the swelling was alleviated, the patient underwent closed reduction and fixation with an Acumed fibular nail and minimally invasive plating osteosynthesis (MIPO), ensuring a smaller incision and minimal soft-tissue dissection. Outcomes: At the 1-year follow-up, the patient had recovered well and had regained satisfactory function in the treated limb. The Kofoed score of the left ankle was 95. Lessons: Based on the experience from this case, the operation can be undertaken safely when the swelling has been alleviated. The minimal invasive technique represents the best approach. Considering the merits and good outcome in this case, we recommend the Acumed fibular nail and MIPO technique for treatment of distal tibial and fibular fractures.

It has been hypothesized that dysregulation of brain-expressed genes is the major predisposing underlying mechanism for autism. This dysregulation may be mediated by differential methylation of CpG sites within gene promoters, which could be candidate biomarkers and used for early clinical screening of autism. A total of 131 pairs of age- and sex-matched autistic and control subjects were recruited in this study. Peripheral blood cells were analyzed. The first five pairs were randomly applied to array-based genome-wide methylation studies. A neuron-specific gene, ENO2, was found to be hypermethylated in the autistic samples. This difference was validated by bisulfite sequencing PCR (BSP). The differential expression of ENO2 gene was further analyzed with RT-qPCR and ELISA. The hypermethylation of ENO2 within the promoter region was confirmed by BSP to be present in 14.5 % (19/131) of the total of the autistic samples. The mean ENO2 RNA level in these 19 autistic samples was reduced by about 70 % relative to that in controls. The average level of ENO2 protein expression in the 19 autistic samples (15.18 ± 3.51 μg/l) was about half of that in the controls (33.86 ± 8.16 μg/l). Conclusion: These findings suggest that reduced ENO2 expression may be a biomarker for a subset of autistic children. Electronic supplementary material The online version of this article (doi:10.1007/s00431-014-2311-9) contains supplementary material, which is available to authorized users.

Rationale The occurrence of peripheral neuropathy associated with non-Hodgkin's lymphoma (NHL) is uncommon. And autoimmunity may play an important role. We report a case of the patient with NHL, has sensorimotor demyelinating polyneuropathy. Patient concerns The patient presented with a 1-month history of progressive numbness at the distal extremities and motor weakness of the lower limbs. Meanwhile, patient also endorsed a painful lump on her right cheek. And then the enlarged cervical and supra clavicular lymph nodes were observed on admission. Biopsy of the lymph nodes showed NHL. Serum IgM antibodies against GM1 and GD1b were also positive. Diagnosis Biopsy of the lymph nodes showed NHL. Serum IgM antibodies against GM1 and GD1b were also positive. Thus, the patience was diagnosed with lymphoma and sensorimotor polyneuropathy. Interventions Patient refused the further treatment. Outcomes After 11-month follow-up, the weakness of bilateral lower limbs worsens. Lessons We have presented a case of NHL involving peripheral polyneuropathy with IgM antibodies against GM1 and GD1b. Patients may initially present with peripheral nerve complications or develop them during the course of lymphoma, even when in remission. This could complicate the diagnosis of peripheral polyneuropathy secondary to NHL.

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca ( 2+) channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.

Antibiotic-loaded polymethylmethacrylate (PMMA) beads are widely used in orthopedic practice for the prevention of infections after open fractures and in the management of osteomyelitis. The use of commercial beads is limited by insufficient flexibility, lack of provision for selection of specific antibiotic, and short drug-release time. Further, the manual procedure for the preparation of PMMA beads is slow, and the products are not uniform in size. Uniformity of the bead size is crucial because the placement of oversized beads place at sites with limited space (e.g., narrow medullary canal) is difficult, and their retrieval from such sites is painful to the patient. To overcome the limitations of commercial beads and manually prepared beads, we developed a simple device for the efficient preparation of antibiotic-loaded PMMA beads of uniform sizes. We describe the device, bead preparation, and the characteristics of the beads prepared using our device, and the preliminary clinical results. The beads obtained using this device were relatively small, had excellent flexibility, and were suitable for implantation in small spaces. The device permits the selection of the antibiotic to be loaded on to the beads. The results of preliminary studies of the beads prepared using our device have been positive, highlighting the need for more large-scale and longitudinal investigations.

The Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease characterized by segmental premature aging. Applying a two-dimensional chromatographic proteomic approach, the 2D Protein Fractionation System (PF2D), we identified 30 differentially expressed proteins in cultured HGPS fibroblasts. We categorized them into five groups: methylation, calcium ion binding, cytoskeleton, duplication, and regulation of apoptosis. Among these 30 proteins, 23 were down-regulated, while seven were up-regulated in HGPS fibroblasts as compared to normal fibroblasts. Three differentially expressed cytoskeleton proteins, vimentin, actin, and tubulin, were validated via Western blotting and characterized by immunostaining that revealed densely thickened bundles and irregular structures. Furthermore in the HGPS cells, the cell cycle G1 phase was elongated and the concentration of free cytosolic calcium was increased, suggesting intracellular retention of calcium. The results that we obtained have implications for understanding the aging process.

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2–CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca2+, but not Na+, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3—the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.

The Hutchinson–Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C ( LMNA ) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.

Problem We have previously determined that long non-coding RNAs (lncRNAs) are differentially expressed in preterm premature rupture of membranes (PPROM) and hypothesized that the collagenolysis ubiquitin–proteasome system may be activated by infection and inflammation. However, direct evidence of the involvement of lncRNAs in transcriptional and posttranscriptional regulation of the infection-triggered alteration of collagen is lacking. Method of study A previously developed mouse model with MHV68 viral infection was assessed to determine whether viral infection may induce differential expression of lncRNAs in mouse placentas and amniotic sacs. Results Differential expression of lncRNAs that are associated with collagen was found in HMV68 viral-infected, compared to non-infected, mouse placentas and amniotic sacs. Differential expression of messenger RNAs (mRNAs) of collagen was also documented. Conclusions Our data demonstrate, for the first time, that viral infection may induce the differential expression of lncRNAs that are associated with collagen. Based on this finding, we propose that lncRNA may have involved in regulating of infection-induced collagen transcription.

Rationale: Clavicle fractures are common, and mostly occur in the midshaft. Methods for operative treatment of midshaft clavicle fractures are evolving, as they improve clinical outcomes compared with traditional conservative management. However, fixation of comminuted midshaft clavicle fractures with bone fragments separated by soft tissue remains a challenge. Patient concerns: Here, we present a case of comminuted midshaft clavicle fracture with a bone fragment separated from the main fracture by soft tissue. Diagnosis: Left comminuted midshaft clavicle fracture. Interventions: We treated this patient with a novel double ligature technique using absorbable suturing. Outcomes: In the past 7 years, we have treated >50 patients with this technique. We have achieved good clinical outcomes with no complications. Lessons: We recommend widespread use of our novel double ligature technique for treating comminuted midshaft clavicle fractures with bone fragments separated by soft tissue.

Activity-dependent neurite outgrowth is a highly complex, regulated process with important implications for neuronal circuit remodeling in development as well as in seizure-induced sprouting in epilepsy. Recent work has linked outgrowth to collapsin response mediator protein 2 (CRMP2), an intracellular phosphoprotein originally identified as axon guidance and growth cone collapse protein. The neurite outgrowth promoting function of CRMP2 is regulated by its phosphorylation state. In this study, depolarization (potassium chloride)-driven activity increased the level of active CRMP2 by decreasing its phosphorylation by GSK3β via a reduction in priming by Cdk5. To determine the contribution of CRMP2 in activity-driven neurite outgrowth, we screened a limited set of compounds for their ability to reduce neurite outgrowth but not modify voltage-gated sodium channel (VGSC) biophysical properties. This led to the identification of (S)-lacosamide ((S)-LCM), a stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Whereas (S)-LCM was ineffective in targeting VGSCs, the presumptive pharmacological targets of (R)-LCM, (S)-LCM was more efficient than (R)-LCM in subverting neurite outgrowth. Biomolecular interaction analyses revealed that (S)-LCM bound to wildtype CRMP2 with low micromolar affinity, similar to (R)-LCM. Through the use of this novel tool, the activity-dependent increase in neurite outgrowth observed following depolarization was characterized to be reliant on CRMP2 function. Knockdown of CRMP2 by siRNA in cortical neurons resulted in reduced CRMP2-dependent neurite outgrowth; incubation with (S)-LCM phenocopied this effect. Other CRMP2-mediated processes were unaffected. (S)-LCM subverted neurite outgrowth not by affecting the canonical CRMP2-tubulin association but rather by impairing the ability of CRMP2 to promote tubulin polymerization, events that are perfunctory for neurite outgrowth. Taken together, these results suggest that changes in the phosphorylation state of CRMP2 are a major contributing factor in activity-dependent regulation of neurite outgrowth.

Problem Spontaneous abortion (SA) occurs before 20 gestational weeks. Approximately, half of recurrent SA has no identifiable cause. No report has yet been investigated the possible involvement of lncRNA in pregnancy loss. Method of Study Sixteen pairs of pregnancies with spontaneous abortions (SA) and induced abortions (IA) were studied. Embryonic sacs and decidua were collected for each pregnancy. A Human LncRNA Array was employed to profile genomewide lncRNAs, which were then validated by RT-PCR. Results Differentially expressed lncRNAs were identified. Biological pathways were categorized into six major groups: infection and inflammation, metabolism, signaling and transcriptional regulation, smooth muscle contraction, cell process, and coagulation. Conclusions Infection and inflammation pathways regulated by lncRNAs were determined as the predominant pathogenetic factors underlying the SA. Finding that antisense lncRNAs have been either up- or down-regulated suggests that they may have both cis- and trans-regulations.

The neuronal ceroid lipofuscinoses (NCLs) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Three typical forms, the infantile (INCL), late-infantile (LINCL), and juvenile (JNCL), are among the most common childhood-onset neurodegenerative disorders. They result from mutations on genes CLN1, CLN2, and CLN3, respectively. We determined that the mutations 223A --G and 451C --T in CLN1, T523-1G --C, and 636 C --T in CLN2, and deletion of a 1.02-kb genomic fragment in CLN3 are the five common mutations for NCL. To offer clinical genetic testing for the NCLs, we have developed simple and quick PCR-based molecular tests for detecting INCL-, LINCL-, and JNCL-affected individuals from 180 NCL families (27 INCL, 76 LINCL, and 77 JNCL). The sensitivity of testing to detect NCL patients among clinically suspected individuals was determined to be 78% (21/27) for INCL, 66% (54/76) for LINCL, and 75% (58/77) for JNCL. When molecular screening for carriers was conducted among the normal siblings or parents of the probands, we identified two carriers out of three individuals tested for INCL, 20/56 (35.7%) carriers for LINCL, and 48/106 (45.3%) carriers for JNCL families. In addition, 5% (9/180) of NCL patients revealed genetic heterogeneity and were reclassified. Seven patients previously diagnosed as having JNCL were now found to carry mutations of CLN2 (5/7) or CLN1 (2/7) and 2 with late-infantile onsets were identified as carrying mutations of CLN1. Our data demonstrate the importance of DNA testing to detect accurately both affected individuals and carriers in NCL families.

Fragile X syndrome is recognized as the most common inherited cause of mental retardation in western countries. The prevalence of the fragile X syndrome in Asian populations is uncertain. We report a multi-institutional collaborative study of molecular screening for the fragile X syndrome from 1,127 Chinese mentally retarded (MR) individuals. We found that 2.8% of the Chinese MR population screened by DNA analysis had the fragile X full mutation. Our screening indicated that the fragile X syndrome prevalence was very close to that of Caucasian subjects. In addition, we found that 62.5% of fragile X chromosomes had a single haplotype for DXS548-FRAXAC1 (21-18 repeats) which was present in only 9.7% of controls. This unique distribution of microsatellite markers flanking the FMR1 CGG repeats suggests that the fragile X syndrome in Chinese populations, as in the Caucasian, may also be derived from founder chromosomes.

Batten disease, the juvenile form of neuronal ceroid lipofuscinosis, is a prevalent neuron degenerative disorder of childhood. A 1.02-kb genomic deletion in the Batten disease gene CLN3 has been determined to be a common mutation. We developed a PCR method to screen for this deletion and tested 43 Batten disease probands. We found 36% (31/86) of Batten disease chromosomes did not carry the 1.02-kb deletion. Of the three heterozygotes for the 1.02-kb deletion, a novel G-to-A missense mutation at nucleotide 1020 of the CLN3 cDNA sequence was found on two of the non-1.02-kb deletion chromosomes. The missense mutation resulted in a substitution of glutamic acid (E) by lysine (K) at position 295 (E295 K). The E295 K mutation causes a change in predicted local protein conformation. This glutamic acid is a highly conserved acidic amino acid, being present in human, mouse, dog and yeast, which suggests it may play an important role in the function of the Batten disease protein.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.

The risk for fragile X "gray-zone" alleles to expand appears to depend on the absence of stabilizing AGGs, which interrupt the CGG repeat region. To characterize such alleles better, we analyzed a series of 101 chromosomes with triplet repeat lengths ranging from 35 to 59 for variations in their AGG interspersion patterns. Among these, 11.9% had 3 AGGs, 59.3% had 2, 24.8% had 1, and 4.0% had 0. An inverse relationship between FMR1 repeat length and the number of interrupting AGGs was observed. Within the range of 35-44 repeats, 98.7% of alleles were found to have a pure CGG repeat length (PCGG) of less than 33. However, among alleles with 45-59 repeats, 50% were found to have 0 or 1 AGG and a PCGG of more than 33. Thus, gray-zone alleles with 45-59 repeats frequently have a long stretch of pure CGGs and thus are more likely to be unstably inherited than alleles with 35-44 repeats. We found length associations of PCGG with 2 flanking microsatellites, DXS548 and FRAXAC1: a PCGGor = 20 was strongly associated with haplotype 20-19, whereas a PCGG20 was more strongly associated with the haplotype 25-21. This result could reflect a founder effect or a generalized instability of CGGs and microsatellites.

FRAXE is a fragile site located at Xq27-8, which contains polymorphic triplet GCC repeats associated with a CpG island. Similar to FRAXA, expansion of the GCC repeats results in an abnormal methylation of the CpG island and is associated with a mild mental retardation syndrome (FRAXE-MR). We surveyed the GCC repeat alleles of FRAXE from 3 populations. A total of 665 X chromosomes including 416 from a New York Euro-American sample (259 normal and 157 with FRAXA mutations), 157 from a Chinese sample (144 normal and 13 FRAXA), and 92 from a Finnish sample (56 normal and 36 FRAXA) were analyzed by polymerase chain reaction. Twenty-seven alleles, ranging from 4 to 39 GCC repeats, were observed. The modal repeat number was 16 in the New York and Finnish samples and accounted for 24% of all the chromosomes tested (162/665). The modal repeat number in the Chinese sample was 18. A founder effect for FRAXA was suggested among the Finnish FRAXA samples in that 75% had the FRAXE 16 repeat allele versus only 30% of controls. Sequencing of the FRAXE region showed no imperfections within the GCC repeat region, such as those commonly seen in FRAXA. The smaller size and limited range of repeats and the lack of imperfections suggests the molecular mechanisms underlying FRAXE triplet mutations may be different from those underlying FRAXA.

The apparent associations between fragile X mutations and nearby microsatellites may reflect both founder effects and microsatellite instability. To gain further insight into their relative contributions, we typed a sample of 56 unrelated control and 37 fragile X chromosomes from an eastern Finnish population for FMR1 CGG repeat lengths, AGG interspersion patterns, DXS548, FRAXAC1, FRAXE and a new polymorphic locus, Alu-L. In the controls, the most common FMR1 allele was 30 repeats with a range of 20 to 47 and a calculated heterozygosity of 88%. A strong founder effect was observed for locus DXS548 with 95% of fragile X chromosomes having the 21 CA repeat (196 bp) allele compared to 17% of controls, while none of the fragile X but 69% of controls had the 20 repeat allele. Although the FRAXAC1 locus is much closer than DXS548 to FMR1 (7 kb vs. 150 kb), there was no significant difference between fragile X and control FRAXAC1 allele distributions. The FRAXE repeat, located 600 kb distal to FMR1, was found to show strong linkage disequilibrium as well. A newly defined polymorphism, Alu-L, located at approximately 40 kb distal to the FMR1 repeat, showed very low polymorphism in the Finnish samples. Analysis of the combined loci DXS548-FRAXAC1-FRAXE showed three founder haplotypes. Haplotype 21-19-16 was found on 27 (75%) of fragile X chromosomes but on none of controls. Three (8.4%) fragile X chromosomes had haplotypes 21-19-15, 21-19-20, and 21-19-25 differing from the common fragile X haplotype only in FRAXE. These could have arisen by recombination or from mutations of FRAXE. A second haplotype 21-18-17 was found in four (11.1%) fragile X chromosomes but only one (1.9%) control. This may represent a more recent founder mutation. A third haplotype 25-21-15, seen in two fragile X chromosomes (5.6%) and one (1.9%) control, was even less common and thus may represent an even more recent mutation or admixture of immigrant types. Analysis of the AGG interspersions within the FMR1 CGG repeat showed that 7/8 premutation chromosomes lacked an AGG whereas all controls had at least one AGG. This supports the hypothesis that the mutation of AGG to CGG leads to repeat instability and mutational expansion.

Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarizationinduced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist.

Rationale Vacuum sealing drainage (VSD) is widely utilized for treating traumatic wounds. Patient concerns It is particularly difficult and time consuming to use in combination with an external fixator. Diagnoses This is because the hardware or pins used for fixation interfere with maintaining a seal, resulting in poor adhesion and subsequent air leakage. Interventions To resolve this problem, we have devised a new method for sealing the wound dressing, while maintaining the required vacuum.When using this technique, a rubber strip is wrapped around each pin in 3 circles outside the plastic drape, and then tightly tied. Outcomes After completing this procedure, a vacuum is obtained, and any air leakage stops. We employed this technique to treat a cohort of patients in our department over a period of two years, and obtained good healing of soft tissue without air leakage, as well as good clinical outcomes. Lessons We have observed that patients treated with this method experienced good clinical outcomes without air leakage, and we recommend its use in treating cases where an external fixation device is present.

The N-type voltage-gated calcium channel (CaV2.2) is a clinically endorsed target in chronic pain treatments. As directly targeting the channel can lead to multiple adverse side effects, targeting modulators of CaV2.2 may prove better. We previously identified ST1-104, a short peptide from the collapsin response mediator protein 2 (CRMP2), which disrupted the CaV2.2-CRMP2 interaction and suppressed a model of HIV-related neuropathy induced by anti-retroviral therapy but not traumatic neuropathy. Here, we report ST2-104 -a peptide wherein the cell-penetrating TAT motif has been supplanted with a homopolyarginine motif, which dose-dependently inhibits the CaV2.2-CRMP2 interaction and inhibits depolarization-evoked Ca(2+) influx in sensory neurons. Ca(2+) influx via activation of vanilloid receptors is not affected by either peptide. Systemic administration of ST2-104 does not affect thermal or tactile nociceptive behavioral changes. Importantly, ST2-104 transiently reduces persistent mechanical hypersensitivity induced by systemic administration of the anti-retroviral drug 2',3'-dideoxycytidine (ddC) and following tibial nerve injury (TNI). Possible mechanistic explanations for the broader efficacy of ST2-104 are discussed.

Autism is a multi-factorial neurodevelopmental disorder. We have investigated the molecular mechanism involved in a Chinese family with autism by a proteomic approach. Antibody chips containing 500 spots of human protein antibodies were used to screen for differentially expressed proteins in the peripheral B lymphocytes between autistic and non-autistic siblings in this family. Four proteins relevant to immuno-pathway, including IKKα that was up-regulated and Tyk2, EIF4G1 and PRKCI that were down-regulated, were identified differentially expressed in autistic versus non-autistic siblings. Western blot analysis and reverse transcription quantitative polymerase chain reaction validated the differential expression of these four proteins. Based on the function of these differentially expressed proteins, relevant studies on immunoglobulin E (IgE) level, nuclear factor kappa B signaling activation and cell cycle were conducted in both autistic and non-autistic children of this family. Considering the fact that the family members were in close contact with natural rubber latex (NRL) and that IgE-mediated cross-reactions could be triggered by Hevea brasiliensis (Hev-b) proteins in NRL, we hypothesize that immune reactions triggered by close contact with NRL might influence the functions of B lymphocytes by altering expression of certain proteins identified in our experiments thus contributing to the occurrence of autism.