A cerebellar model of timing and prediction in the control of reaching.
A simplified model of the cerebellum was developed to explore its potential for adaptive, predictive control based on delayed feedback information. An abstract representation of a single Purkinje cell with multistable properties was interfaced, using a formalized premotor network, with a simulated single degree-of-freedom limb. The limb actuator was a nonlinear spring-mass system based on the nonlinear velocity dependence of the stretch reflex. By including realistic mossy fiber signals, as well as realistic conduction delays in afferent and efferent pathways, the model allowed the investigation of timing and predictive processes relevant to cerebellar involvement in the control of movement. The model regulates movement by learning to react in an anticipatory fashion to sensory feedback. Learning depends on training information generated from corrective movements and uses a temporally asymmetric form of plasticity for the parallel fiber synapses on Purkinje cells.


Meta-analysis: association between wrist posture and carpal tunnel syndrome among workers.
Carpal tunnel syndrome (CTS) is a common work-related peripheral neuropathy. In addition to grip force and repetitive hand exertions, wrist posture (hyperextension and hyperflexion) may be a risk factor for CTS among workers. However, findings of studies evaluating the relationship between wrist posture and CTS are inconsistent. The purpose of this paper was to conduct a meta-analysis of existing studies to evaluate the evidence of the relationship between wrist posture at work and risk of CTS. PubMed and Google Scholar were searched to identify relevant studies published between 1980 and 2012. The following search terms were used: "work related", "carpal tunnel syndrome", "wrist posture", and "epidemiology". The studies defined wrist posture as the deviation of the wrist in extension or flexion from a neutral wrist posture. Relative risk (RR) of individual studies for postural risk was pooled to evaluate the overall risk of wrist posture on CTS. Nine studies met the inclusion criteria. All were cross-sectional or case-control designs and relied on self-report or observer's estimates for wrist posture assessment. The pooled RR of work-related CTS increased with increasing hours of exposure to wrist deviation or extension/flexion [RR = 2.01; 95% confidence interval (CI): 1.646-2.43; p < 0.01: Shore-adjusted 95% CI: 1.32-2.97]. We found evidence that prolonged exposure to non-neutral wrist postures is associated with a twofold increased risk for CTS compared with low hours of exposure to non-neutral wrist postures. Workplace interventions to prevent CTS should incorporate training and engineering interventions that reduce sustained non-neutral wrist postures.


The influence of neighbor relatedness on multilevel selection in the Great Lakes sea rocket.
Natural selection can operate at the individual and group level in natural populations. This study investigates the ecological factors that determine the relative importance of individual versus group selection. In particular, it determines how the relatedness of interacting neighbors influences multilevel natural selection in a population of the Great Lakes sea rocket. Focal plants were grown in groups of siblings, groups of plants that were themselves siblings but unrelated to the focal plants, and groups of plants with mixed genotypes. Significant group selection on plant size was observed only when the neighbors were siblings but not when they were unrelated. In sibling groups, individuals with heavier stems had higher fitness, and individuals growing with heavier but shorter neighbors also had higher fitness. Thus, individual and group selection on stem weight operated in the same direction. The detection of group selection in sibling groups can be attributed in part to an increased opportunity for group selection in these groups since sibling groups differed more from one another than the other group types. In addition, the quality of the selective environment in sibling groups may have differed from that for the other group types. Group selection was therefore more prevalent in the most genetically structured sample, in which responses to group selection are also most likely to occur.


Kinetic study of anti-viral ribavirin uptake mediated by hCNT3 and hENT1 in Xenopus laevis oocytes.
Transport across the cell membrane is crucial in drug delivery. However, the process is complicated because nucleoside derivatives that are commonly used as anti-viral drugs are transported through two different types of specific transporters: concentrative transporters and equilibrative transporters. Cross-disciplinary approaches involving both biological experiments and theoretical considerations are therefore necessary to study the transport of nucleoside analogues such as ribavirin. Here we constructed an experimental model system using the Xenopus laevis oocyte that expressed examples of both types of transporters: human concentrative nucleoside transporter 3 and human equilibrative transporter 1. We also performed a kinetic study. Experimental results showed that the transport of ribavirin could be reduced by inhibiting one of the two types of transporters, which seems to be counterintuitive. We therefore designed a simple mathematical model of the dynamics of ribavirin uptake and analyzed the model behaviors using a numerical simulation. The theoretical results reproduced the experimentally observed phenomena and suggested a possible mechanism for the process. Based on this mechanism, we predicted some potential methods for the effective uptake of ribavirin from a dynamics point of view.


Mortality and neurodevelopmental outcomes of acute bacterial meningitis in children aged <5 years in Pakistan.
Significant neurodevelopmental sequelae are known to occur after acute bacterial meningitis (ABM). This study determined the burden of such sequelae in Pakistani children aged <5 years to guide policies for Haemophilus influenzae type b (Hib) and pneumococcal vaccination. Cases of ABM were recruited from hospital-based surveillance and assigned to 1 of 3 etiologic groups (Hib, Streptococcus pneumoniae, or unknown etiology). Two age-matched controls were recruited for each case. Six months after enrollment, each case underwent neurologic history and examination, neurodevelopmental evaluation, and neurophysiological hearing test. Controls were assessed in parallel. Of 188 cases, 64 (34%) died. Mortality among subgroups were 7 (27%), 14 (28%), and 43 (39%) for Hib, Streptococcus pneumoniae, and unknown etiology, respectively. Eighty cases and 160 controls completed the assessments. Sequelae among cases included developmental delay (37%), motor deficit (31%), hearing impairment (18.5%), epilepsy (14%), and vision impairment (14%). Sequelae were higher after pneumococcal meningitis (19, 73%) compared with Hib meningitis (8, 53%). Compared with controls, cases were at significantly higher risk for all sequelae (P < .0001). ABM causes a substantial long-term burden of poor neurodevelopmental outcomes. Hib and pneumococcal vaccines are very effective interventions to prevent meningitis and its disabling sequelae.


B Lymphocytes as Targets of the Immunomodulatory Properties of Human Amniotic Mesenchymal Stromal Cells.
Mesenchymal stromal cells (MSC) from the amniotic membrane of human term placenta (hAMSC), and the conditioned medium generated from their culture (CM-hAMSC) offer significant tools for their use in regenerative medicine mainly due to their immunomodulatory properties. Interestingly, hAMSC and their CM have been successfully exploited in preclinical disease models of inflammatory and autoimmune diseases where depletion or modulation of B cells have been indicated as an effective treatment, such as inflammatory bowel disease, lung fibrosis, would healing, collagen-induced arthritis, and multiple sclerosis. While the interactions between hAMSC or CM-hAMSC and T lymphocytes, monocytes, dendritic cells, and macrophages has been extensively explored, how they affect B lymphocytes remains unclear. Considering that B cells are key players in the adaptive immune response and are a central component of different diseases, in this study we investigated the in vitro properties of hAMSC and CM-hAMSC on B cells. We provide evidence that both hAMSC and CM-hAMSC strongly suppressed CpG-activated B-cell proliferation. Moreover, CM-hAMSC blocked B-cell differentiation, with an increase of the proportion of mature B cells, and a reduction of antibody secreting cell formation. We observed the strong inhibition of B cell terminal differentiation into CD138+ plasma cells, as further shown by a significant decrease of the expression of interferon regulatory factor 4 (IRF-4), PR/SET domain 1(PRDM1), and X-box binding protein 1 (XBP-1) genes. Our results point out that the mechanism by which CM-hAMSC impacts B cell proliferation and differentiation is mediated by secreted factors, and prostanoids are partially involved in these actions. Factors contained in the CM-hAMSC decreased the CpG-uptake sensors (CD205, CD14, and TLR9), suggesting that B cell stimulation was affected early on. CM-hAMSC also decreased the expression of interleukin-1 receptor-associated kinase (IRAK)-4, consequently inhibiting the entire CpG-induced downstream signaling pathway. Overall, these findings add insight into the mechanism of action of hAMSC and CM-hAMSC and are useful to better design their potential therapeutic application in B-cell mediated diseases.


Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform.
We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (microTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 microm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 microl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.


The asymptomatic ocular surface reservoir of coronavirus disease-19 in infants and retinopathy of prematurity screening during the pandemic.
This study aimed to evaluate the incidence of conjunctival and pharyngeal swab sample positivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic preterm and term infants. We aimed to detect asymptomatic carrier potential in infants. Patients screened for retinopathy of prematurity (ROP) at our clinic between January and June 2021 were recruited for this study. For all study cases, the previous history of possible exposure or contact with SARS-CoV-2, previous history of coronavirus disease-19 (COVID-19), or contact with any COVID-19 case was excluded. None of the patients showed signs of COVID-19 during sample collection. Pharyngeal and conjunctival swab samples were collected before the ophthalmic examination. Nucleic acid isolation from the samples was performed using an automated system. The presence of SARS-CoV-2 RNA in the samples was screened using a real-time polymerase chain reaction kit, and the positive samples were re-evaluated for the variant virus. Among the 127 patients with a median age of 40 weeks (range: 34-86) of postmenstrual age, positivity for SARS-CoV-2 RNA in the pharyngeal and conjunctival samples was 5/127 (3.93%) and 3/127 (2.36%), respectively. Isolated conjunctival positivity was not observed in any of the patients, and all three patients were positive for both conjunctiva and pharynx. Asymptomatic infants may be a reservoir for SARS-CoV-2, and conjunctival infection in infants may be a source of virus transmission. Since ROP screening cannot be postponed during the pandemic, caution should be exercised to prevent the spread of the disease.


Congenital neurodevelopmental diagnoses and an intensive care unit: defining a population.
To identify and describe the population of children with congenital or perinatally acquired neurodevelopmental diagnoses in a pediatric intensive care unit and to assess the nature and extent of their utilization of critical care resources. Twelve-month, inception cohort study. Intensive care unit at an urban, tertiary care pediatric hospital. All pediatric intensive care unit admissions were screened for preexisting neurodevelopmental diagnoses. Computerized and chart-based medical records were reviewed for demographic, clinical, and outcome data. A total of 309 children with congenital neurodevelopmental diagnoses accounted for 427 pediatric intensive care unit admissions. This represented 23% of the total 1,820 admissions in 1 yr. Trisomy 21 was the most identifiable developmental abnormality (n = 25, 8%). Eighty-five percent of the children were cared for at home before hospitalization. A total of 220 of the admissions (52%) demonstrated a preexisting technology dependence. Fewer children admitted from the home-care setting had tracheostomies or were ventilator dependent. The majority of admissions were scheduled surgical admissions (45%) or for management of acute respiratory illness (26%). Of the patients with preexisting tracheostomy, nonrespiratory conditions accounted for 70% of acute admitting diagnoses. Two hundred twenty-three of the admissions (52%) required noninvasive or transtracheal ventilatory support, yet the length of stay and mortality rate were consistent with those reported in other general pediatric intensive care unit populations. The average and median length of stay were 5.4 and 2.0 days, respectively. Mortality rate was 3%. Technology support needs at discharge increased significantly from admission for enterostomy support (p =.008) and mechanical ventilation (p =.008). Children with congenital or perinatally acquired neurodevelopmental diagnoses represented nearly one quarter of all pediatric intensive care unit admissions at a tertiary academic center. This population has substantial ongoing medical needs, requiring utilization of intensive care resources. More rigorous investigations are needed to determine the effect of this burgeoning population in pediatric critical care, to optimize their care, and to meet the comprehensive needs of their families.


Subclinical renal injury induced by transient cyclosporine exposure is associated with salt-sensitive hypertension.
Cyclosporine use is highly associated with the development of salt-sensitive hypertension. We hypothesized that subtle renal injury induced by cyclosporine could lead to salt sensitivity. Cyclosporine nephropathy was induced by treatment for 4 weeks with cyclosporine (15 mg/kg/day) on a low sodium (0.05%) diet, followed by stopping cyclosporine and placement on a high sodium (4%) diet for 4 additional weeks. Control groups included a group treated with cyclosporine (15 mg/kg/day) on a normal salt diet in which nephropathy does not develop, and a vehicle-treated group. A fourth group received half-dose of cyclosporine (8 mg/kg/day) on a low sodium diet, which results in mild nephropathy. Biopsies were obtained at the end of the cyclosporine administration (4 week) and at sacrifice (8 week), and blood pressure and renal function were measured. Rats treated with cyclosporine for 4 weeks on a low sodium diet developed classic features of tubulointerstitial disease and arteriolopathy; these changes were absent in the cyclosporine/normal salt group and in the vehicle group. At 4 weeks, all groups were switched to a high salt diet; only the rats with nephropathy developed hypertension. The degree of hypertension correlated closely with the degree of tubulointerstitial injury (r=0.85) and with the severity of the arteriolopathy (r=0.9) (p<0.0.1). Importantly, renal function (creatinine clearance) was normal in all groups at 8 weeks, documenting that the hypertension could not be attributed to cyclosporine-mediated alterations in glomerular filtration rate (GFR). One mechanism by which cyclosporine induces hypertension is the induction of subtle renal microvascular and tubulointerstitial disease. This mechanism is not dependent on GFR and may persist even after the cyclosporine is discontinued.


Progression from hormone dependence to autonomy and angiogenesis in mouse mammary tumours.
The transplantable pregnancy-dependent mammary tumour (TPDMT-4), the related hormone-dependent (TPDMT-4EP) and autonomous (T4-0I320 and T4-0I96) subline tumours, and the mammary glands from DDD mice were compared for angiogenic activity on the rabbit cornea by tissue implantation. The TPDMT-4EP tumour was established by serially transplanting TPDMT-4 tumour fragments in oestradiol plus progesterone treated mice. The T4-0I320 and T4-0I96 tumours directly derived from the TPDMT-4 and TPDMT-4EP tumours, respectively. Angiogenic activity was graded by macroscopic and microscopic examinations into 3 classes; negative, partial and complete angiogenesis. These tumours were comparable to mammary glands in activity and induced complete angiogenesis in only 15-23% of the implants. However, when partial and complete responses were combined as positive angiogenesis, TPDMT-4, T4-0I320, TPDMT-4EP and T4-0I96 tumour implants were angiogenic in 25, 29, 42 and 54%, respectively. The T4-0I96 tumour was significantly more angiogenic than the parent tumour but this was not so for the TPDMT-4EP tumour. Spontaneous C3H mouse mammary tumours, human gliomas from nude mice, rat Walker 256 carcinomas and rabbit VX-2 tumours induced complete angiogenesis in 54, 63, 59 and 92% of the implants, respectively. The results suggest that the TPDMT-4 tumour is unique in being weakly angiogenic and able to progress toward greater autonomy with or without augmented angiogenic activity in different conditions.


Abscisic Acid Inhibits Rice Protein Phosphatase PP45 via H2O2 and Relieves Repression of the Ca2+/CaM-Dependent Protein Kinase DMI3.
In plants, Ca2+/calmodulin-dependent protein kinase (CCaMK) is a positive regulator of abscisic acid (ABA) responses, including root growth, antioxidant defense, and tolerance of both water stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, we show a direct interaction between DMI3 (Doesn't Make Infections 3), a rice (Oryza sativa) CCaMK and PP45, a type 2C protein phosphatase in rice (PP2C). This interaction involves the CaM binding domain of DMI3 and the PP2C domain of PP45. In the absence of ABA, PP45 directly inactivates DMI3 by dephosphorylating Thr-263 in DMI3. However, in the presence of ABA, ABA-induced H2O2 production by the NADPH oxidases RbohB/E inhibits the activity of PP45 not only by inhibiting the expression of PP45 but also by oxidizing Cys-350 and Cys-428 residues to form PP45 intermolecular dimers. ABA-induced oxidation of Cys-350 and Cys-428 in PP45 blocked the interaction between PP45 and DMI3 and substantially prevented PP45-mediated inhibition in DMI3 activity. Genetic analysis indicated that PP45 is an important negative regulator of ABA signaling. These results reveal important pathways for the inhibition of DMI3 under the basal state and for its ABA-induced activation in rice.


The molecular basis of C6 deficiency in the western Cape, South Africa.
Deficiency of the sixth component of human complement (C6) has been reported in a number of families from the western Cape, South Africa. Meningococcal disease is endemic in the Cape and almost all pedigrees of total C6 deficiency (C6Q0) have been ascertained because of recurrent disease. We have sequenced the expressed exons of the C6 gene from selected cases and have found three molecular defects leading to total deficiency: 879delG, which is the common defect in the Cape and hitherto unreported, and 1195delC and 1936delG, which have been previously reported in African-Americans. We also show that the 879delG and 1195delC defects are associated with characteristic C6/C7 region DNA marker haplotypes, although small variations were observed. The 1936delG defect was observed only once in the Cape, but its associated haplotype could be deduced. The data from the haplotypes indicate that these three molecular defects account for the defects in all the 38 unrelated C6Q0 individuals we have studied from the Cape. We have also observed the 879delG defect in two Dutch C6-deficient kindreds, but the 879delG defect in the Cape probably did not come from The Netherlands.


Relationships Between Childhood Interpersonal Trauma, Religious Coping, Post-traumatic Stress Symptoms, and Resilience.
Positive and negative religious coping strategies have been linked to symptom trajectories following adult interpersonal trauma. However, the interactions between childhood interpersonal trauma, religious coping, and psychological outcomes are less clear. This study examined whether aspects of religious coping moderated the associations between cumulative childhood interpersonal trauma and mental health outcomes, such as post-traumatic stress symptoms (PTSS) and resilience. Participants included 525 undergraduates from two universities (Mage = 20.04, SD = 1.71; range = 18-24; 57.7% White; 82.1% female). In both the positive and negative religious coping models, cumulative childhood interpersonal trauma was related to PTSS (b = 6.66; b = 6.10, respectively). While positive religious coping was not associated with PTSS (b = .01), it was linked to resilience (b = .69). Negative religious coping was significantly related to PTSS (b = .75) but not resilience (b = -.20). No significant interactions were identified between aspects of religious coping and cumulative childhood interpersonal trauma. While religious coping was directly related to both positive and negative mental health outcomes, it may not be associated with the relationships between childhood interpersonal trauma exposure and clinical outcomes. Such findings offer valuable information on malleable factors that may contribute to adaptive and maladaptive functioning following childhood adversity.


Comparative genomics of small RNA regulatory pathway components in vector mosquitoes.
Small RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways. The Ae. aegypti, An. gambiae and Cx. pipiens genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. Ae. aegypti and Cx. pipiens have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome. Analysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, Ae. aegypti and Cx. pipiens, are evolving faster than those of the malaria vector An. gambiae and D. melanogaster. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1) key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. Implications for arbovirus vector competence in mosquitoes are discussed.


Clinical and in vitro resistance to GS-9669, a thumb site II nonnucleoside inhibitor of the hepatitis C virus NS5B polymerase.
Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5× the 50% effective concentration [EC50]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20× the EC50). During the phase I clinical study, substitutions at NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669 monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423 at baseline. Consistent with the low resistance level at this position, three patients with M423I or M423V at baseline achieved >2-log10 reductions of HCV RNA when treated with 100 mg BID or with 500 mg QD or BID of GS-9669. The fourth patient, who had the M423V substitution at baseline, had a 4.4-log10 reduction of HCV RNA with 500 mg BID of GS-9669. Phenotypic analyses demonstrated that the viral isolates with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and lomibuvir (VX-222) but are not cross-resistant to other classes of HCV inhibitors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01431898.).


Down-staging of hepatocellular carcinoma via external-beam radiotherapy with subsequent liver transplantation: a case report.
Despite the widespread use of locoregional therapies [radiofrequency ablation and transarterial chemoembolization (TACE)], there is currently a lack of high-quality evidence supporting their use for hepatocellular carcinoma (HCC) in patients on the liver transplantation (LT) waiting list or requiring down-staging. Radiotherapy has rarely been used in this setting and has usually been in the form of more complex and less accessible techniques such as proton-beam and stereotactic body radiation therapy. Only 1 report describes the use of conventional 3-dimensional conformal external-beam radiotherapy (cEBRT) techniques as neoadjuvant or down-staging therapy for patients who are LT candidates. This report describes the use of cEBRT in a 52-year-old hepatitis C-positive man with cirrhosis. A 40-mm right lobe HCC was treated initially with TACE while he was on the waiting list. The lesion progressed beyond transplant criteria (76 mm). Conventional external-beam radiotherapy (EBRT) was used (54 Gy in 27 fractions) to down-stage the lesion. EBRT was well tolerated and resulted in a complete radiological response with no arterial enhancement of the lesion for a total of 16 months. Subsequent LT and a review of the explant demonstrated complete histological necrosis of the lesion. This report provides the first description of complete histological necrosis of HCC through the use of cEBRT techniques as down-staging/neoadjuvant therapy before LT. Because of its potential efficacy, accessibility, tolerability, noninvasive and outpatient nature, and ability to treat lesions adjacent to vessels and biliary structures, further trials examining the efficacy of cEBRT versus other neoadjuvant techniques are urgently required.


Ultrastructural identification of cells involved in the healing of intramembranous bone grafts in both the presence and absence of demineralised intramembranous bone matrix.
Alveolar bone defects are conditions that impede the progress of orthodontic treatment. This study compared the mechanics of the healing of autogenous intramembranous (IM) bone grafts and grafts comprising a mixture of IM and demineralised bone matrix of autogenous intramembranous origin (DBMIM), in an attempt to determine the reliability of each material. Thirty-two New Zealand white rabbits had a single defect created in their skull. Sixteen were grafted with IM bone alone (Group I: autogenous IM), and the other 16 had a combined graft of composite IM sandwiched between two layers of DBMIM (Group II: composite IM-DBMIM). A third group (Group III) of eight rabbits each had two defects created in their skull; one defect was left empty (A: passive control) and the other filled with rabbit-skin collagen (B: active control). In Groups I and II, inflammatory cells were found to be present on Days 1 and 2 of tissue retrieval. The appearance of the mesenchymal cells and preosteoblasts, osteoblasts and osteocytes was earlier (Day 3) in Group II than in Group I (Day 5). In both groups, preosteoblasts, osteoblasts and osteocytes were observed with no cartilage at the intermediate stage. In conclusion, autogenous IM bone grafts and IM bone grafts in the presence of DBMIM healed through an osteogenic ossification route.


Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.
Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.


Inhibiting the anaphase promoting complex/cyclosome induces a metaphase arrest and cell death in multiple myeloma cells.
The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase involved in cell cycle. During the metaphase-anaphase transition the APC/C is activated by Cdc20. The aim of this study is to elucidate the importance and therapeutic potential of APC/C and its co-activator Cdc20 in multiple myeloma (MM). Gene expression analysis revealed that Cdc20 was expressed at higher levels in gene expression-based high-risk MM patients. Moreover, high Cdc20 expression correlated with poor prognosis. Treatment of human myeloma cell lines with proTAME, an APC/C inhibitor, resulted in an accumulation of APC/CCdc20 substrate cyclin B1 and an accumulation of cells in metaphase. Moreover we observed a significant dose-dependent decrease in viability and increase in apoptosis in MM cells upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. A similar metaphase arrest and induction of apoptosis were obtained with specific knockdown of Cdc20. In addition, we demonstrated the accumulation of Bim was partially responsible for the observed cell death. Combining proTAME with another APC/C inhibitor apcin or the alkylating agent melphalan resulted in enhanced anti-MM activity. This study suggests that the APC/C and its co-activator Cdc20 could be a new and promising target especially in high-risk MM patients.


Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism.
Bidirectional communication between oocytes and the companion granulosa cells is essential for the development and functions of both compartments. Oocytes are deficient in their ability to transport certain amino acids and in carrying out glycolysis and cholesterol biosynthesis. Cumulus cells must provide them with the specific amino acids and the products in these metabolic pathways. Oocytes control metabolic activities in cumulus cells by promoting the expression of genes in cumulus cells encoding specific amino acid transporters and enzymes essential for the oocyte-deficient metabolic processes. Hence oocytes outsource metabolic functions to cumulus cells to compensate for oocyte metabolic deficiencies. Oocyte control of granulosa cell metabolism may also participate in regulating the rate of follicular development in coordination with endocrine, paracrine, and autocrine signals. Oocytes influence granulosa cell development mainly by secretion of paracrine factors, although juxtacrine signals probably also participate. Key oocyte-derived paracrine factors include growth differentiation factor 9, bone morphogenetic protein 15, and fibroblast growth factor 8B.


[Defective forearm pseudoarthrosis with a neglected elbow dislocation].
Neglected elbow dislocations, rarely seen in the developed countries, are very attractive due to the difficulties of treatment and diversity of prognosis. A 32 year-old woman was admitted with chronic elbow dislocation and defective forearm pseudoarthrosis due to an accident four months prior to admission. Open reduction and distraction arthroplasty using hinged external fixator for the elbow, open reduction and internal fixation with plate-screws with fibula allograft for the forearm was performed. The elbow was stable and had 110 degrees and -15 degrees of active flexion and extension respectively on the ninth month of follow-up. Both radius and ulna were united.


Clearing-factor lipase in obese hyperglycaemic mice (ob-ob).
1. Clearing-factor lipase was assayed in acetone-ether-dried powders of heart and epididymal fat-pads of lean and genetically obese mice (ob/ob). In both tissues the enzyme activity in the adult was higher in the obese mice. 2. In heart the enzyme activity was unchanged from 8 to 48 weeks of age in lean mice, but in obese mice it increased between 8 and 12 weeks of age and remained elevated. 3. Starvation produced changes in the heart clearing-factor lipase activity in obese, but not lean, mice. 4. The clearing-factor lipase activity of epididymal fat-pads decreased rapidly during 24h starvation in both lean and obese mice, but the activity in the obese mice remained higher than that in lean mice. 5. Plasma triglyceride and cholesterol concentrations were determined in both lean and obese mice. Triglyceride concentrations were not greatly different, but the obese mice were hypercholesterolaemic. Plasma cholesterol concentrations were not correlated with changes in clearing-factor lipase activity.


Genome-scale fluxes predicted under the guidance of enzyme abundance using a novel hyper-cube shrink algorithm.
One of the long-expected goals of genome-scale metabolic modelling is to evaluate the influence of the perturbed enzymes on flux distribution. Both ordinary differential equation (ODE) models and constraint-based models, like Flux balance analysis (FBA), lack the capacity to perform metabolic control analysis (MCA) for large-scale networks. In this study, we developed a hyper-cube shrink algorithm (HCSA) to incorporate the enzymatic properties into the FBA model by introducing a pseudo reaction V constrained by enzymatic parameters. Our algorithm uses the enzymatic information quantitatively rather than qualitatively. We first demonstrate the concept by applying HCSA to a simple three-node network, whereby we obtained a good correlation between flux and enzyme abundance. We then validate its prediction by comparison with ODE and with a synthetic network producing voilacein and analogues in Saccharomyces cerevisiae. We show that HCSA can mimic the state-state results of ODE. Finally, we show its capability of predicting the flux distribution in genome-scale networks by applying it to sporulation in yeast. We show the ability of HCSA to operate without biomass flux and perform MCA to determine rate-limiting reactions. Algorithm was implemented by Matlab and C ++. The code is available at https://github.com/kekegg/HCSA. firstname.lastname@example.com or email@example.com. Supplementary data are available at Bioinformatics online.


Modality-specific segregation of input to ant mushroom bodies.
The mushroom bodies are central brain neuropils involved in the control of complex behavior. In ants, the mushroom bodies are relatively large compared to those of honey bees, whereas the optic lobes of ants are considerably smaller. The general morphology of ant mushroom bodies is similar to that of honey bees. As in other Hymenoptera, the main input region of the mushroom bodies, the calyx, is subdivided into three compartments: the lip, the collar, and the basal ring. In many ant species this compartmentalization is not obvious and can only be visualized using neuronal tracers. The lip region receives antennal input and is large in all ant species. It appears to be composed of at least two different regions that have not yet been characterized in detail. The collar is large in other Hymenoptera, yet in ant workers it varies in size and is always much smaller than the lip region. The collar receives visual input and is relatively larger in males, which generally are more dependant on vision than are workers. The basal ring receives input from both the optic and antennal lobes. In one ant tribe, the Ponerini, the collar region appears to have changed its position, but based on afferent input it appears to be homologous to the hymenopteran collar. Generally, the composition of the mushroom body calyx correlates with the living conditions of ants, reflecting the great importance of olfaction and the lesser and more variable significance of vision for workers of the observed ant species.


Can we make smart choices between OLS and contaminated IV methods?
In the outcomes research and comparative effectiveness research literature, there are strong cautionary tales on the use of instrumental variables (IVs) that may influence the newly initiated to shun this premier tool for casual inference without properly weighing their advantages. It has been recommended that IV methods should be avoided if the instrument is not econometrically perfect. The fact that IVs can produce better results than naïve regression, even in nonideal circumstances, remains underappreciated. In this paper, we propose a diagnostic criterion and related software that can be used by an applied researcher to determine the plausible superiority of IV over an ordinary least squares (OLS) estimator, which does not address the endogeneity of a covariate in question. Given a reasonable lower bound for the bias arising out of an OLS estimator, the researcher can use our proposed diagnostic tool to confirm whether the IV at hand can produce a better estimate (i.e., with lower mean square error) of the true effect parameter than the OLS, without knowing the true level of contamination in the IV.


Surgical management of chronic venous insufficiency.
Chronic venous insufficiency is a pathologic condition of the skin and subcutaneous tissues in the lower extremity caused by stasis of the blood flow. Incompetency or failure of the venous valves results in reflux and ambulatory venous hypertension, which is more severe with deep than with superficial venous incompetency. Superficial chronic venous insufficiency (varicose veins) is effectively managed with ligation and stripping of incompetent perforator and superficial veins to restore normal venous physiology. Deep chronic venous insufficiency (postphlebitic leg) presents a widespread pathologic disorder that is refractory to surgical correction. Adjunctive surgical measures such as removal of incompetent perforators or superficial veins to lessen local stasis or skin grafting of ulcers are often indicated in selected cases. The underlying chronic venous insufficiency requires management with elastic compression, elevation of the legs, and exercise for best results.


Use of rich BHI medium instead of synthetic TMH medium for gene regulation study in Yersinia pestis.
This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the β-Galactosidase enzyme assay system. When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP. The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.


Classification of Large Cellular Populations and Discovery of Rare Cells Using Single Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.
Cell-to-cell variability and functional heterogeneity are integral features of multicellular organisms. Chemical classification of cells into cell type is important for understanding cellular specialization as well as organismal function and organization. Assays to elucidate these chemical variations are best performed with single cell samples because tissue homogenates average the biochemical composition of many different cells and oftentimes include extracellular components. Several single cell microanalysis techniques have been developed but tend to be low throughput or require preselection of molecular probes that limit the information obtained. Mass spectrometry (MS) is an untargeted, multiplexed, and sensitive analytical method that is well-suited for studying chemically complex individual cells that have low analyte content. In this work, populations of cells from the rat pituitary, the rat pancreatic islets of Langerhans, and from the Aplysia californica nervous system, are classified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) MS by their peptide content. Cells were dispersed onto a microscope slide to generate a sample where hundreds to thousands of cells were separately located. Optical imaging was used to determine the cell coordinates on the slide, and these locations were used to automate the MS measurements to targeted cells. Principal component analysis was used to classify cellular subpopulations. The method was modified to focus on the signals described by the lower principal components to explore rare cells having a unique peptide content. This approach efficiently uncovers and classifies cellular subtypes as well as discovers rare cells from large cellular populations.


Central Granular Cell Odontogenic Tumor: Report of a Case with CBCT Features.
Central granular cell odontogenic tumor) CGCOT) of the jaw is an exceedingly rare benign odontogenic neoplasm with 35 reported cases in the literature. Among these, very few studies have focused on the cone-beam CT features of CGCOT. Here, we report a case of an asymptomatic CGCOT in a 16-year-old girl and focus on the cone-beam CT features. Only 36 cases of this lesion, including this one, have been reported so far. The case presented is of special importance due to the young age of the patient, the posterior location of the lesion and the multilocular pattern in the cone beam CT images.


Ecological partitioning among parapatric cryptic species.
Geographic range differences among species may result from differences in their physiological tolerances. In the intertidal zone, marine and terrestrial environments intersect to create a unique habitat, across which physiological tolerance strongly influences range. Traits to cope with environmental extremes are particularly important here because many species live near their physiological limits and environmental gradients can be steep. The snail Melampus bidentatus occurs in coastal salt marshes in the western Atlantic and the Gulf of Mexico. We used sequence data from one mitochondrial (COI) and two nuclear markers (histone H3 and a mitochondrial carrier protein, MCP) to identify three cryptic species within this broad-ranging nominal species, two of which have partially overlapping geographic ranges. High genetic diversity, low population structure, and high levels of migration within these two overlapping species suggest that historical range limitations do not entirely explain their different ranges. To identify microhabitat differences between these two species, we modelled their distributions using data from both marine and terrestrial environments. Although temperature was the largest factor setting range limits, other environmental components explained features of the ranges that temperature alone could not. In particular, the interaction of precipitation and salinity likely sets physiological limits that lead to range differences between these two cryptic species. This suggests that the response to climatic change in these snails will be mediated by changes to multiple environmental factors, and not just to temperature alone.


Isotope encephalography in the management of acute herpesvirus encephalitis.
Two patients with Herpesvirus hominis (herpes simplex) encephalitis were investigated by serial isotope encephalograms using technetium-99m pertechnetate. In the first case the diagnosis was made by brain biopsy, and by successful tissue culture, and was confirmed by a significant rise in antibody titre, but in the second the laboratory evidence was exclusively serological. Necrotizing encephalitis was presumed in both cases because the illness was grave and focal signs developed in conjunction with radiological and electroencephalographic evidence of circumscribed lesions of the hemisphere. The emergence of new lesions in the brain scan at a time of clinical remission was found to be a warning of impending relapse. Such lesions had to be distinguished from areas of increasing uptake of isotope due only to local change in vascular permeability. Decay of EEG activity together with clinical signs of focal abnormality over an area of isotope concentration indicated a necrotizing lesion. Cytosine arabinoside (cytarabine), an alternative to idoxuridine as an antiviral drug, was used in both cases. A third patient, suspected in life of having a degenerative encephalopathy, was found at necropsy to have necrotizing encephalitis. Herpes infection though probable was not confirmed. The most severe lesions were in the frontal and temporal lobes, which had shown increased uptake of technetium in the encephalogram performed six weeks before her death.


Comparison of local anesthetics as bronchodilator aerosols.
Lidocaine aerosols have been ineffective at preventing airway constriction provoked by non-reflex stimuli. To determine if there were any advantages to using more potent amide-type local anesthetics or local anesthetics from other chemical classes, the authors compared the protection afforded by pretreatment with aerosols of lidocaine, bupivacaine, hexylcaine, and procaine against a subsequent prolonged challenge (5 min) with 10% citric acid aerosols in barbiturate-anesthetized Basenji-Greyhound dogs. The local anesthetics were administered as aerosols (4% solutions) for 10 min immediately preceding the citric acid challenge. In control experiments, that employed pretreatment with aerosols of isotonic saline rather than local anesthetics, the citric acid challenge aerosol increased pulmonary resistance by an average of 3.3 +/- 0.8 (+/- SE) cmH2O X 1(-1) X s during the first 15-min postchallenge, and reduced dynamic compliance by one-third during this time. None of the local anesthetics prevented citric-acid-induced changes in pulmonary mechanics. The average increases in pulmonary resistance (0-15 min postchallenge) elicited by citric acid aerosols in dogs pretreated with lidocaine, hexylcaine, bupivacaine, and procaine were 2.1 +/- 0.6, 2.1 +/- 0.6, 3.2 +/- 1.3, and 3.3 +/- 1.0 cmH2O X 1(-1) X s, respectively. The authors concluded that local anesthetic aerosols have little or no effect against other than reflex bronchoconstriction, and none of those tested were more effective than lidocaine.


Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct.
2019-nCoV is the causative agent of the serious, still ongoing, worldwide coronavirus disease (COVID-19) pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease (HRV3C) cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA immobilized metal ion affinity chromatography (IMAC) purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.


Medical emergency rescue in disaster: the international emergency response to the Haiyan typhoon in Philippines.
Following Typhoon Haiyan, the World Health Organization (WHO) has been supporting the Government of the Philippines in coordinating the incoming relief supplies from more than 30 international humanitarian health organizations. During the 10 days in Abuyong, Philippines, the Chinese medical rescue team consisting of 50 experts specialized in clinical medicine and disease prevention and control action was taken including, medical treatment, environmental disinfection and health education. A total of 1,831 cases and 2,144 outpatients were treated, blood tests, B-ultrasound, electrocardiogram (ECG) and other laboratory examinations were carried out for more than 615 patients; a cumulative 90,000 square meters in external environment were disinfected, and more than 500 health education materials were handed out. Besides, measures of purifying drinking water, and rebuilding the local hospital have also been carried out. The international emergency response to the Haiyan typhoon in Philippines contributed to reconstruct the local disaster health system by the activities from international medical emergency rescue. To improve the capacity of international medical emergency rescue in disaster, the special project of foreign medical emergency rescue should be set in countries' medical emergency rescue, and disaster emergency medical rescue should be reserved as a conventional capacity.


Incidence of Clostridium botulinum in crabmeat from the blue crab.
The incidence of Clostridium botulinum in fresh crabmeat of blue crab was six out of 986 samples; in pasteurized crabmeat one sample out of 1,000 contained the organism.


Microfilament organization and total actin content are decreased in hybrids derived from the fusion of HeLa cells with human fibroblasts.
The organization of the microfilaments and the actin content of matched pairs of tumorigenic and non-tumorigenic HeLa/fibroblast hybrid cells was compared. Each pair consisted of a hybrid cell line with suppressed tumorigenicity and a segregant tumorigenic cell line derived from it. The tumorigenic HeLa parent cell line and a non-tumorigenic human fibroblast line were also included in the comparison. Microfilament organization of the cell lines was assessed by fluorescence microscopy using NBD-phallacidin, a probe that specifically binds to actin filaments. The re-expression of tumorigenicity is associated with a loss of microfilament organization. The actin content, as measured by the DNase I inhibition assay, was significantly lower in the tumorigenic hybrids and the HeLa parent than in the non-tumorigenic cells. The comparison was significant when the actin concentration was expressed either per cell or per protein. Despite this reduced level of total actin in tumorigenic cells, the ratio of monomeric to total actin remained constant in all cell lines tested.


Abnormal functional connectivity of the reward network is associated with social communication impairments in autism spectrum disorder: A large-scale multi-site resting-state fMRI study.
The social motivation hypothesis proposes that the social deficits of autism spectrum disorder (ASD) are related to reward system dysfunction. However, functional connectivity (FC) patterns of the reward network in ASD have not been systematically explored yet. The reward network was defined as eight regions of interest (ROIs) per hemisphere, including the nucleus accumbens (NAc), caudate, putamen, anterior cingulate cortex (ACC), ventromedial prefrontal cortex (vmPFC), orbitofrontal cortex (OFC), amygdala, and insula. We computed both the ROI-wise resting-state FC and seed-based whole-brain FC in 298 ASD participants and 348 typically developing (TD) controls from the Autism Brain Imaging Data Exchange I dataset. Two-sample t-test was applied to obtain the aberrant FCs. Then, the association between aberrant FCs and clinical symptoms was assessed with Pearson's correlation or Spearman's correlation. In addition, Neurosynth Image Decoder was used to generate word clouds verifying the cognitive functions of the aberrant pathways. Furthermore, a three-way multivariate analysis of variance (MANOVA) was conducted to examine the effects of gender, subtype and age on the atypical FCs. For the within network analysis, the left ACC showed weaker FCs with both the right amygdala and left NAc in ASD compared with TD, which were negatively correlated with the Autism Diagnostic Observation Schedule (ADOS) total scores and Social Responsiveness Scale (SRS) total scores respectively. For the whole-brain analysis, weaker FC (i.e., FC between the left vmPFC and left calcarine gyrus, and between the right vmPFC and left precuneus) accompanied by stronger FC (i.e., FC between the left caudate and right insula) were exhibited in ASD relative to TD, which were positively associated with the SRS motivation scores. Additionally, we detected the main effect of age on FC between the left vmPFC and left calcarine gyrus, of subtype on FC between the right vmPFC and left precuneus, of age and age-by-gender interaction on FC between the left caudate and right insula. Our findings highlight the crucial role of abnormal FC patterns of the reward network in the core social deficits of ASD, which have the potential to reveal new biomarkers for ASD.


[Vaccination of cattle against respiratory disease caused by virus infection (author's transl)].
Studies on the epizootiology of respiratory viruses in herds of cattle showed that infection by bovine respiratory syncytial (BRS) virus was a major cause of bovine respiratory disease in calves. Moreover, of IBR virus infection associated with respiratory symptoms were observed in the Netherlands since the Spring of 1974. Outbreaks of IBR virus infections occur throughout the Netherlands today. Initially, outbreaks ran a course marked by the typical symptoms of haemorhagic rhinitis, observed particularly in cows. Today, IBR virus infections have become enzootic on several farms, the majority of infections in young cattle being accompanied by respiratory symptoms that are identified as being due to IBR virus infection. Other virus infections that may cause respiratory disease, parainfluenza virus 3, adeno viruses and bovine virus diarrhoea (BVD) virus, were found to be enzootic in most herds in the Netherlands. However, there is no definite correlation between the occurrence of these infections and clinical symptoms. The use of vaccines against these virus infections is not advisable in the present situation. A vaccine against BRS virus infections is commerically available. Immunization by this vaccine against the pulmonary symptoms of BRS virus infection should be verified by a field trial. Unvaccinated control groups of calves should be included. In the experience of the present author, the immunity produced by this vaccine cannot prevent infection by the field strain of BRS virus. Postvaccination reactions were observed within seven days after vaccination with this vaccine in some cases. These were attributable to non-cytopathogenic strains of BVD virus, contaminating some batches of this commerical vaccine.


Action pattern and substrate specificity of the hyaluronan lyase from group B streptococci.
The hyaluronan lyase of group B streptococci rapidly cleaves hyaluronan by an elimination mechanism to yield the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose. Additionally, it has been shown that the enzyme has limited specificity for achondroitin sulphate and cleaves the chain at unsulphated sites [Baker,Yu, Morrison, Averett and Pritchard (1997) Biochem. J. 327,65-71]. In the present extension of that study it was found that 6-sulphated regions of chondroitin sulphate are also susceptible to cleavage by this hyaluronan lyase. Of the four 6- and/or 4-sulphated tetrasaccharides which can be isolated from testicular hyaluronidase digests of chondroitin sulphate, only those two tetrasaccharides with a6-sulphated disaccharide at the reducing end were cleaved. From thisand other data, a model is proposed for the cleavage specificity of hyaluronan lyase on a chondroitin sulphate. Evidence is presented in support of an action pattern for hyaluronan lyase which involves aninitial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharide. Since the on lyoligosaccharides which tend to accumulate in near-complete digests of hyaluronan are unsaturated, it is argued that the processive cleavage occurs from the non-reducing to the reducing end of a hyaluronan chain. This detailed knowledge of substrate specificity contributes to our understanding of the enzyme's role in Group B streptococcal pathogenesis. In addition, the hyaluronan lyase may find application in sequence studies of chondroitin sulphates.


Segregation of nociceptive and non-nociceptive networks in the squirrel monkey somatosensory thalamus.
The somatosensory thalamus (here we examine neurons in the caudal cutaneous portion of ventral posterior lateral nucleus, VPL) is composed of a somatotopic arrangement of anteroposteriorly oriented rods. Each rod is a collection of neurons with homogeneous properties that relay sensory information to specific cortical columns. We developed a multi-electrode recording technique, using fixed-geometry four-tip electrodes that allow simultaneous recordings from small populations of neurons (4-11), in a approximately 150 x 150 x 150 micrometer(3) volume of brain tissue (i.e., the approximate diameter of rods) and study of their spatiotemporal interactions. Due to the fixed geometry of the four-tip electrodes, the relative locations of these neurons can be determined, and due to the simultaneity of the recordings, their spike-timing coordination can be calculated. With this method, we demonstrate the existence of two distinct functional networks: nociceptive and non-nociceptive networks. The population dynamics of these two types of networks are different: cross-correlations in each type of network were different in direction and strength, were a function of the distance between neurons, had an opponent organization for nociceptive networks and a non-opponent organization for non-nociceptive networks, and rapidly changed under different stimulus conditions independent of changes in firing rates. A simple neural network model mimicked these physiological findings, demonstrating the necessity of inhibitory interneurons and different amounts of afferent input synchronization. Based on these results, we conclude that the somatosensory thalamus is composed of two modules, nociceptive and non-nociceptive rods, and that the response dynamics differences between these modules are due to spatiotemporal differences of their afferent inputs.


A daily glass of red wine and lifestyle changes do not affect arterial blood pressure and heart rate in patients with carotid arteriosclerosis after 4 and 20 weeks.
Regular consumption of small amounts of red wine improves blood lipids. However, there is concern whether this beneficial effect might be counterbalanced by an increase in blood pressure (BP) and heart rate (HR), which are risk factors for cerebro-cardiovascular disease. In particular, we studied whether regular consumption of red wine with and without lifestyle changes (LC; healthy diet and physical activity advice) results in an increase in BP and HR. A prospective, unblinded randomized trial was performed in 108 patients (67% men) with carotid atherosclerosis documented by ultrasound, a mean BP of 122/79 mm Hg and a mean HR of 71 bpm at inclusion in the study. Sixty-eight percent were known and treated hypertensives. The mean 24-hour BP at baseline was 122/79 mm Hg. Half of the study participants, the control group, was seen by a nurse at baseline, after 4 and after 20 weeks, and was instructed not to change their eating and physical activity habits. In the other half, a dietician performed five sessions of 30 min each (at baseline, after 1 week and after 2, 3 and 4 weeks) giving advice on healthy eating based on a Mediterranean diet and physical exercise. The recommendations given were the following: 5 portions of fruit/vegetables per day, a diet low in absolute fat, a preference of vegetable oil (olive or rapeseed oil), whole-grain products, poultry, low-fat dairy products, 1 fat and 1 lean fish meal per week, reduced consumption of red meat, and avoidance of pork, ready-made meals, sugar and excessive salt intake. In addition, regular consumption of 1 bar of dark chocolate (25 g, >70% of cacao), 1-2 tomatoes, and 3-5 walnuts as well as at least 30 min of moderate daily physical activity were recommended. Within these two groups, half of the patients were randomized either to avoid alcohol completely or to drink 100 ml (women) or 200 ml of red wine (men) daily. Neither LC nor red wine had an effect on the mean systolic and diastolic 24-hour BP and HR after 4 and 20 weeks, as analyzed by general linear modeling. No difference was found for diurnal and nocturnal values. The possible beneficial effect of regular consumption of small amounts of red wine is not counterbalanced in the long term by an increase in the mean BP or HR in mainly normotensive and well-treated hypertensive patients with carotid atherosclerosis, neither in the patients given healthy lifestyle advice nor in those with a standard lifestyle. Yet, we remain cautious about actively advice patients to drink alcohol regularly given the well-known risks.


Acute kidney injury secondary to severe delayed haemolysis in intravenous artesunate use for severe malaria.
The use of artemisinin derivatives has been recommended by the WHO guidelines in malaria treatment largely due to its rapid parasite clearance and safety profile. This case report details the development of delayed haemolysis and subsequent severe acute kidney injury (AKI) 13 days after commencing intravenous artesunate treatment for malaria in an Australian returned traveller. Delayed haemolysis may be an under-recognised complication following artesunate use and if severe, can be complicated by AKI. Therefore, close patient follow-up following treatment is required to ensure prompt recognition of this phenomenon.


Relaxation times and rheology in dense athermal suspensions.
We study the jamming transition in a model of elastic particles under shear at zero temperature. The key quantity is the relaxation time τ which is obtained by stopping the shearing and letting energy and pressure decay to zero. At many different densities and initial shear rates we do several such relaxations to determine the average τ. We establish that τ diverges with the same exponent as the viscosity and determine another exponent from the relation between τ and the coordination number. Though most of the simulations are done for the model with dissipation due to the motion of particles relative to an affinely shearing substrate, we also examine a model, where the dissipation is instead due to velocity differences of disks in contact, and confirm that the above-mentioned exponent is the same for these two models. We also consider finite size effects on both τ and the coordination number.


The statistical challenge of analysing changes in dual energy computed tomography (DECT) urate volumes in people with gout.
Dual energy computed tomography (DECT) allows direct visualization of monosodium urate crystal deposition in gout. However, DECT urate volume data are often highly skewed (mostly small volumes with the remainder considerably larger), making statistical analyses challenging in longitudinal research. The aim of this study was to explore the ability of various analysis methods to normalise DECT urate volume data and determine change in DECT urate volumes over time. Simulated datasets containing baseline and year 1 DECT urate volumes for 100 people with gout were created from two randomised controlled trials. Five methods were used to transform the DECT urate volume data prior to analysis: log-transformation, Box-Cox transformation, log(X-(min(X)-1)) transformation; inverse hyperbolic sine transformation, and rank order. Linear regression analyses were undertaken to determine the change in DECT urate volume between baseline and year 1. Cohen's d were calculated as a measure of effect size for each data treatment method. These analyses were then tested in a validation clinical trial dataset containing baseline and year 1 DECT urate volumes from 91 people with gout. No data treatment method successfully normalised the distribution of DECT urate volumes. For both simulated and validation data sets, significant reductions in DECT urate volumes were observed between baseline and Year 1 across all data treatment methods and there were no significant differences in Cohen's d effect sizes. Normalising highly skewed DECT urate volume data is challenging. Adopting commonly used transformation techniques may not significantly improve the ability to determine differences in measures of central tendency when comparing the change in DECT urate volumes over time.


COVID-19 and Guillain-Barré Syndrome: A Case Report and Review of Literature.
During the recent coronavirus disease 2019 (COVID-19) outbreak in Northern Italy, we observed a 57-year-old man developing acute motor-sensory axonal neuropathy, a variant of Guillain-Barré syndrome (GBS), 12 days after severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Similarly to other bacterial and viral infections, dysregulation of the immune system due to post-infectious mechanisms, such as the molecular mimicry, could lead to an indirect damage of the peripheral nervous system related to SARS-CoV-2. GBS causes motor dysfunctions that are not easily recognizable in non-neurological settings or in patients requiring ventilatory assistance. Several reports also suggested that GBS and Miller Fisher syndrome (MFS) could be neurological complications of COVID-19. Therefore, we performed a review of the 29 articles so far published, describing 33 GBS cases and five MFS cases associated with SARS-CoV-2 infection. We recommend awareness of this rare, but treatable, neurological syndrome, which may also determine a sudden and otherwise unexplained respiratory deterioration in COVID-19 patients.


A modified Delphi approach to develop a trial protocol for antibiotic de-escalation in patients with suspected sepsis.
Early administration of antibiotics in sepsis is associated with improved patient outcomes, but safe and generalizable approaches to de-escalate or discontinue antibiotics after suspected sepsis events are unknown. We used a modified Delphi approach to identify safety criteria for an opt-out protocol to guide de-escalation or discontinuation of antibiotic therapy after 72 hours in non-ICU patients with suspected sepsis. An expert panel with expertise in antimicrobial stewardship and hospital epidemiology rated 48 unique criteria across 3 electronic survey rating tools. Criteria were rated primarily based on their impact on patient safety and feasibility for extraction from electronic health record review. The 48 unique criteria were rated by anonymous electronic survey tools, and the results were fed back to the expert panel participants. Consensus was achieved to either retain or remove each criterion. After 3 rounds, 22 unique criteria remained as part of the opt-out safety checklist. These criteria included high-risk comorbidities, signs of severe illness, lack of cultures during sepsis work-up or antibiotic use prior to blood cultures, or ongoing signs and symptoms of infection. The modified Delphi approach is a useful method to achieve expert-level consensus in the absence of evidence suifficient to provide validated guidance. The Delphi approach allowed for flexibility in development of an opt-out trial protocol for sepsis antibiotic de-escalation. The utility of this protocol should be evaluated in a randomized controlled trial.


[Distorted Freiburg Speech Test].
In 107 patients with different types of hearing loss (air conduction, sensorineural and combined) we ascertained the monaural discrimination loss of monosyllables with the help of the normal and distorted Freiburg speech test. It was evident that the quotient obtained from the measured discrimination is a very good and useful measure to characterise the ability to analyse the peripheral hearing system. Compared with normal hearing individuals and patients with air conduction hearing loss or combined loss of hearing, persons with perception hearing loss show a much greater and more divergent quotient. The discrimination of distorted speech depends more on age than that of normal speech; however, it is considerably reduced from the sixth decade of life onwards. The distortion test offers the expertising the possibility of improved assessment of speech discrimination by workers in a noisy environment in case of unfavourable hearing conditions, but it is also of considerable importance in fitting hearing aids and in the diagnosis of acoustic tumours.


Safety and Success Rates of Excimer Laser Sheath-Assisted Retrieval of Embedded Inferior Vena Cava Filters.
Despite historically high rates of use, most inferior vena cava (IVC) filters are not retrieved. The US Food and Drug Administration safety communications recommended retrieval when the IVC filter is no longer indicated out of concern for filter-related complications. However, failure rates are high when using standard techniques for retrieval of long-dwelling filters, and until recently, there have been no devices approved for retrieval of embedded IVC filters. To evaluate the safety and success of excimer laser sheath-assisted retrieval of embedded IVC filters. A retrospective, multicenter, clinical cohort study of excimer laser sheath-assisted IVC filter retrievals from 7 US sites was conducted between March 1, 2012, and February 28, 2021, among 265 patients who underwent IVC filter retrieval using the laser. Patients were substratified between a high-volume single center and a multicenter data set. A blinded physician committee adjudicated reported complications and their association with use of the laser. Retrieval of IVC filters using excimer laser sheath. The primary safety end point was device-related major complication rate (Society of Interventional Radiology categories C to F, which included any adverse event associated with morbidity or disability that increases the level of care, results in hospital admission, or substantially lengthens the hospital stay). The primary success end point was technical success of IVC filter retrieval. The primary end points were compared with literature-derived, meta-analysis-suggested target performance goals. The single-center experience included 139 participants (mean [SD] age, 52 [16] years; 78 female participants [56.1%]), and the multicenter experience included 126 participants (mean [SD] age, 52 [16] years; 75 female participants [59.5%]). The device-related major complication rate was 2.9% (4 of 139; 95% CI, 0.8%-7.2%; P = .001) for the single-center experience and 4.0% (5 of 126; 95% CI, 1.3%-9.0%; P = .01) for the multicenter experience, both of which were significantly lower than the primary safety performance goal (10%). No major complications were considered to be definitively associated with use of the laser. The technical success rate was 95.7% (133 of 139; 95% CI, 90.8%-98.4%; P = .007) for the single-center experience and 95.2% (120 of 126; 95% CI, 89.9%-98.2%; P = .02) for the multicenter experience, both of which were significantly higher than the primary performance goal (89.4%). This cohort study demonstrated high technical success and low complication rates of excimer laser sheath-assisted retrieval of embedded IVC filters in centers with variable case volume and experience, which suggests a wide applicability of the technique with proper training. The excimer laser sheath offers physicians a valuable tool for retrieval of challenging embedded IVC filters.


The positive influence of sense of control on physical, behavioral, and psychosocial health in older adults: An outcome-wide approach.
Accumulating research indicates robust associations between sense of control and salutary health and well-being outcomes. However, whether change in sense of control is associated with subsequent outcomes has been under-evaluated. Participants (N = 12,998) were from the Health and Retirement Study-a diverse, nationally representative, and longitudinal sample of U.S. adults aged >50 years. We examined how increase in sense of control (from t0:2006/2008 to t1: 2010/2012) was associated with better outcomes on 35 indicators of: physical-, behavioral-, and psychosocial-health (t2:2014/2016). We used multiple logistic-, linear-, and generalized-linear regression models and controlled for sociodemographic characteristics, personality traits, sense of control, and all outcomes in the pre-baseline wave (t0:2006/2008). During the 4-year follow-up, people in the highest (vs. lowest) quartile of sense of control, conditional on prior sense of control, had reduced risk of mortality and improved physical-health outcomes (lower risk of: stroke, lung disease, physical limitations, cognitive impairment, chronic pain and higher self-rated health). Sense of control was related to better health-behaviors (increased physical activity, reduced sleep problems), higher psychological well-being (positive affect, life satisfaction, optimism, purpose, personal-, health-, financial-mastery), lower psychological distress (depression, hopelessness, negative affect, perceived constraints), decreased loneliness, and increased contact with friends. Sense of control was unrelated to other physical health indicators (diabetes, hypertension, heart disease, cancer, arthritis, overweight/obesity), health behaviors (binge drinking, smoking), and social factors (living with spouse/partner, frequency of contact with children and other family). These findings underscore the importance of sense of control as a potential intervention target for fostering physical-, behavioral-, and psychosocial-health.


Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.
Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.


Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis.
This paper aimed at investigating the expression and methylation profiles of SOX2, a gene coding for the stem cell-related transcription factor SOX2, in endometrial carcinomas. By methylation-specific polymerase chain reaction (MS-PCR), the methylation status of SOX2 promoter region in 72 endometrial carcinomas and 12 normal endometrial samples was examined. Methylated allele was found in 37.5% (27/72) of endometrial carcinomas but only in 8.3% (1/12) of normal endometrial, significantly more frequent in cancers (P = .0472). SOX2 mRNA level was significantly reduced in endometrial carcinoma compared with nonneoplastic endometrium (P = .045). A significant correlation between SOX2 mRNA expression and hypermethylation of SOX2 was found (P = .024). Hypermethylation of SOX2 tended to be more frequently found in type II serous or clear cell adenocarcinoma. SOX2 methylation was also significantly correlated with shorter survival of patients (P = .046). In conclusion, epigenetic mechanisms may play a crucial role on the transcriptional regulation of SOX2 and loss of SOX2 expression may be related to endometrial carcinogenesis.


Influence of Obesity on Breast Density Reduction by Omega-3 Fatty Acids: Evidence from a Randomized Clinical Trial.
Preclinical data indicate that omega-3 fatty acids (n-3FA) potentiate the chemopreventive effect of the antiestrogen (AE) tamoxifen against mammary carcinogenesis. The role of n-3FA in breast cancer prevention in humans is controversial. Preclinical and epidemiologic data suggest that n-3FA may be preferentially protective in obese subjects. To directly test the protective effect of n-3FA against breast cancer, we conducted a 2-year, open-label randomized clinical trial in 266 healthy postmenopausal women (50% normal weight, 30% overweight, 20% obese) with high breast density (BD; ≥25%) detected on their routine screening mammograms. Eligible women were randomized to one of the following five groups (i) no treatment, control; (ii) raloxifene 60 mg; (iii) raloxifene 30 mg; (iv) n-3FA lovaza 4 g; and (v) lovaza 4 g plus raloxifene 30 mg. The 2-year change in BD, a validated biomarker of breast cancer risk, was the primary endpoint of the study. In subset analysis, we tested the prespecified hypothesis that body mass index (BMI) influences the relationship between plasma n-3FA on BD. While none of the interventions affected BD in the intention-to-treat analysis, increase in plasma DHA was associated with a decrease in absolute breast density but only in participants with BMI >29. Our results suggest that obese women may preferentially experience breast cancer risk reduction from n-3FA administration.


MicroRNA-378 attenuates myocardial fibrosis by inhibiting MAPK/ERK pathway.
To elucidate the role of microRNA-378-containing microvesicles (MVs) in the process of myocardial fibrosis and its underlying mechanism. In vivo chronic myocardial fibrosis (MF) model in rats was established by aortic coarctation method. MicroRNA-378 mimic or inhibitor was injected into the rat tail vein at day 3 after the aortic coarctation. Two weeks later, rats were sacrificed for collecting myocardium. MVs were isolated from rat cardiomyocytes and further verified by detecting the expression level of its marker CD63. Expression levels of fibrosis-related indicators and microRNA-378 in MVs were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. After induction with transforming growth factor-β1 (TGF-β1) in primary rat cardiomyocytes for different time points, expression levels of fibrosis-related indicators and microRNA-378 were also accessed. Changes in mitogen-activated protein kinase (MAPK) pathway were observed during the process of MF by qRT-PCR and Western blot. Expression levels of microRNA-378-containing MVs decreased, and the MAPK pathway was activated during the process of MF, which further aggravated MF. MicroRNA-378-containing MVs alleviate myocardial fibrosis through inhibiting the phosphorylation of MAPK.


Creating higher titer lentivirus with caffeine.
The use of lentiviral vectors extends from the laboratory, where they are used for basic studies in virology and as gene transfer vectors gene delivery, to the clinic, where clinical trials using these vectors for gene therapy are currently underway. Lentiviral vectors are useful for gene transfer because they have a large cloning capacity and a broad tropism. Although procedures for lentiviral vector production have been standardized, simple methods to create higher titer virus during production would have extensive and important applications for both research and clinical use. Here we present a simple and inexpensive method to increase the titer by 3- to 8-fold for both integration-competent lentivirus and integration-deficient lentivirus. This is achieved during standard lentiviral production by the addition of caffeine to a final concentration of 2-4 mM. We find that sodium butyrate, a histone deacetylase inhibitor shown previously to increase viral titer, works only ∼50% as well as caffeine. We also show that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) inhibitor NU7026 can also increase viral titer, but that the combination of caffeine and NU7026 is not more effective than caffeine alone. We show that the time course of caffeine treatment is important in achieving a higher titer virus, and is most effective when caffeine is present from 17 to 41 hr posttransfection. Last, although caffeine increases lentiviral vector titer, it has the opposite effect on the titer of adeno-associated virus type 2 vector. Together, these results provide a novel, simple, and inexpensive way to significantly increase the titer of lentiviral vectors.


Antioxidants and Health-Beneficial Nutrients in Fruits of Eighteen Cucurbita Cultivars: Analysis of Diversity and Dietary Implications.
Aging is accompanied by gradual accumulation of molecular damage within cells in response to oxidative stress resulting from adverse environmental factors, inappropriate lifestyle, and numerous diseases. Adequate antioxidant intake is a key factor of proper diet. The study aimed to assess the antioxidant/antiradical capacities of Cucurbita fruits (18 cultivars of the species: C. maxima Duch., C. moschata Duch., C. pepo L., and C. ficifolia Bouché) grown in central Europe. The analyses were based on the FRAP (ferric reducing antioxidant power), CUPRAC (cupric ion reducing antioxidant capacity), and DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assays. The content of phenolic compounds and β-carotene was evaluated with HPLC (high performance liquid chromatography), while the main macro- and micronutrients by ICP-OES (inductively coupled plasma mass spectrometry). The results revealed high intraspecies variability within the Cucurbita genus. The Japanese 'Kogigu' fruits were distinguished as extraordinary sources of phenolic compounds, including syringic and protocatechuic acids, catechin, and kaempferol. Another popular cultivar 'Hokkaido' exhibited the highest antioxidant and antiradical capacities. Most of the fruits proved to be rich sources of zinc and copper. The obtained data are discussed in the context of optimized nutrition of the elderly and suggest that Cucurbita fruits should become daily components of their diet.


Differential expression of miRNAs in response to topping in flue-cured tobacco (Nicotiana tabacum) roots.
Topping is an important cultivating measure for flue-cured tobacco, and many genes had been found to be differentially expressed in response to topping. But it is still unclear how these genes are regulated. MiRNAs play a critical role in post-transcriptional gene regulation, so we sequenced two sRNA libraries from tobacco roots before and after topping, with a view to exploring transcriptional differences in miRNAs. Two sRNA libraries were generated from tobacco roots before and after topping. Solexa high-throughput sequencing of tobacco small RNAs revealed a total of 12,104,207 and 11,292,018 reads representing 3,633,398 and 3,084,102 distinct sequences before and after topping. The expressions of 136 conserved miRNAs (belonging to 32 families) and 126 new miRNAs (belonging to 77 families) were determined. There were three major conserved miRNAs families (nta-miR156, nta-miR172 and nta-miR171) and two major new miRNAs families (nta-miRn2 and nta-miRn26). All of these identified miRNAs can be folded into characteristic miRNA stem-loop secondary hairpin structures, and qRT-PCR was adopted to validate and measure the expression of miRNAs. Putative targets were identified for 133 out of 136 conserved miRNAs and 126 new miRNAs. Of these miRNAs whose targets had been identified, the miRNAs which change markedly (>2 folds) belong to 53 families and their targets have different biological functions including development, response to stress, response to hormone, N metabolism, C metabolism, signal transduction, nucleic acid metabolism and other metabolism. Some interesting targets for miRNAs had been determined. The differential expression profiles of miRNAs were shown in flue-cured tobacco roots before and after topping, which can be expected to regulate transcripts distinctly involved in response to topping. Further identification of these differentially expressed miRNAs and their targets would allow better understanding of the regulatory mechanisms for flue-cured tobacco response to topping.


Detection of the production of oxygen-centered free radicals by human neutrophils using spin trapping techniques: a critical perspective.
Human neutrophils generate oxygen reduction products as a consequence of membrane interactions with a number of stimuli. One oxygen-centered free radical (superoxide) has been unequivocally shown to result from this "respiratory burst," and some experimental evidence for another (hydroxyl radical) has been published, although debate remains as to its significance. The role of phagocyte-derived free radicals in microbicidal and tumoricidal activity as well as tissue damage at sites of inflammation has been the focus of extensive investigation in recent years. Of the techniques available to study free radical generation in biological systems, spin trapping has emerged as a powerful tool for detection and identification of these reactive species, owing in part to its ability to measure production of free radicals inside the phagosome. However, interpretation of resulting spectra is extremely complex and filled with pitfalls and limitations. In this communication we review spin-trapping techniques and discuss the application of this system to the identification of free radicals resulting from stimulation of human neutrophils. Criteria are established which are necessary for definitive identification of superoxide and hydroxyl radical when employing this technology. In this context a critical perspective of previous studies of neutrophil-derived free radicals is offered.


Standardization of Plant Microbiome Studies: Which Proportion of the Microbiota is Really Harvested?
Studies in plant-microbiome currently use diverse protocols, making their comparison difficult and biased. Research in human microbiome have faced similar challenges, but the scientific community proposed various recommendations which could also be applied to phytobiome studies. Here, we addressed the isolation of plant microbiota through apple carposphere and lettuce root microbiome. We demonstrated that the fraction of the culturable epiphytic microbiota harvested by a single wash might only represent one-third of the residing microbiota harvested after four successive washes. In addition, we observed important variability between the efficiency of washing protocols (up to 1.6-fold difference for apple and 1.9 for lettuce). QIIME2 analysis of 16S rRNA gene, showed a significant difference of the alpha and beta diversity between protocols in both cases. The abundance of 76 taxa was significantly different between protocols used for apple. In both cases, differences between protocols disappeared when sequences of the four washes were pooled. Hence, pooling the four successive washes increased the alpha diversity for apple in comparison to a single wash. These results underline the interest of repeated washing to leverage abundance of microbial cells harvested from plant epiphytic microbiota whatever the washing protocols, thus minimizing bias.


Ferulic acid supplements abrogate oxidative impairments in liver and testis in the streptozotocin-diabetic rat.
The primary objective of this study was to assess the efficacy of ferulic acid (FA), a phenolic antioxidant, in ameliorating oxidative stress in the testis and liver of diabetic pubertal rats. Male (6 wk old) rats were rendered diabetic by an acute dose (60 mg/kg body weight, intraperitoneal) of streptozotocin (STZ) and were given oral supplementation of FA (50 mg/kg body weight/d on alternate days) for 4 weeks. The protective efficacy of FA was assessed by measuring markers of oxidative stress in the testis and liver along with the effect of stress on lipid profile in serum/testis. Terminally, the testis (cytosol and mitochondria) of STZ-administered rats exhibited a marked elevation in the status of lipid peroxidation and enhanced reactive oxygen species (ROS) production compared to the non-diabetic controls. FA treatment completely normalized the oxidative impairments in the testis. Further, STZ-induced depletion of reduced glutathione (GSH) and elevated protein carbonyl content in the testis were restored to normalcy by FA treatment. The protective effects of FA were also discernible in the testis in terms of restoration of activities of various antioxidant enzymes in the diabetic rats. Furthermore, STZ-induced oxidative impairments in the liver were also abrogated significantly by FA treatment. STZ-induced perturbations in serum and testicular lipid profiles in the diabetic rats were also significantly attenuated by FA treatment. Collectively, these results indicate that oral supplementation of FA can significantly mitigate diabetes-associated oxidative impairments in the testis as well as in the liver and suggests the efficacy of FA as a complementary therapeutic agent in the management of diabetes-associated oxidative stress-mediated complications.


Quantitative in vitro assay for "corncob" formation.
The interaction of Bacterionema matruchotii with strains of Streptococcus sanguis produces a structure which morphologically resembles a corncob. To determine the specific bacterial surface receptors involved in the interaction, we developed a quantitative assay. The assay consisted of mixing saline suspensions of [CH(3)-(3)H]thymidine-labeled streptococci and B. matruchotii, incubating at 37 degrees C for 2 h, and filtering the mixture through a 5-mum polycarbonate membrane filter. The free cocci and filaments passed through the filter, but the corncobs were retained. Estimates of the number of corncobs formed were obtained by quantitating the radioactivity retained on the membranes relative to that of controls of streptococci alone. Although saturation of the Bacterionema occurred at a ratio of streptococci to Bacterionema of 10:1 (Klett units), a 2:1 ratio was chosen because of the increased sensitivity of the assay at this ratio. The percentage of streptococci binding at this ratio was 18.6 +/- 8.1 (standard deviation). All five Bacterionema strains tested formed corncobs; in contrast, only three strains of S. sanguis were positive. These were serotype 1 strains which had localized surface "fuzz." Although scanning electron microscopic observations revealed an almost random distribution of cocci along the filament surface, transmission electron microscopy revealed that the streptococci were attached to the Bacterionema by the surface fuzz. No differences in corncob formation were observed in sodium phosphate buffer, pH 6 to 8, at phosphate concentrations ranging from 0.005 to 0.05 M. Concentrations of NaCl or KCl up to 0.25 M did not affect corncob formation, and low concentrations of CaCl(2) increased corncob formation slightly, whereas MgCl(2), ethylenediaminetetraacetic acid, and citrate buffers reduced the number of streptococci binding to the filaments. These results suggest that divalent cations may play a role in this process.


[Uses of fresh herbs with antioxidant effect and prospect for population ecology reconstruction in Macau].
The average life expectancy in Macau is ranking the second in the world, the consumption of fresh medicinal plants is a profound culture in Macau. The paper focus on the distribution of the antioxidant herbs, a comprehensive investigation and analysis the amount of the plant resources was carried out. The antioxidant activity of alcohol extracts was determined by using the DPPH method, and six kinds fresh herbs with high antioxidant free radical activity were screened out. Reference to adult daily dose of vitamin C, it is calculated that the daily dose amount of fresh herbs is less than 200 g. For the expected shortage of resources and the ecological status of Macau, we give some suggestions of herbal introduction in population ecology reconstruction.