Copy number variation of KIR genes influences HIV-1 control.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Does the physical disector method provide an accurate estimation of sensory neuron number in rat dorsal root ganglia?

The physical disector is a method of choice for estimating unbiased neuron numbers; nevertheless, calibration is needed to evaluate each counting method. The validity of this method can be assessed by comparing the estimated cell number with the true number determined by a direct counting method in serial sections. We reconstructed a 1/5 of rat lumbar dorsal root ganglia taken from two experimental conditions. From each ganglion, images of 200 adjacent semi-thin sections were used to reconstruct a volumetric dataset (stack of voxels). On these stacks the number of sensory neurons was estimated and counted respectively by physical disector and direct counting methods. Also, using the coordinates of nuclei from the direct counting, we simulate, by a Matlab program, disector pairs separated by increasing distances in a ganglion model. The comparison between the results of these approaches clearly demonstrates that the physical disector method provides a valid and reliable estimate of the number of sensory neurons only when the distance between the consecutive disector pairs is 60 microm or smaller. In these conditions the size of error between the results of physical disector and direct counting does not exceed 6%. In contrast when the distance between two pairs is larger than 60 microm (70-200 microm) the size of error increases rapidly to 27%. We conclude that the physical dissector method provides a reliable estimate of the number of rat sensory neurons only when the separating distance between the consecutive dissector pairs is no larger than 60 microm.

Patterns of pain-free response in 497 cases of classic trigeminal neuralgia treated with Gamma Knife surgery and followed up for least 1 year.

Object The goal of this study was to establish whether clear patterns of initial pain freedom could be identified when treating patients with classic trigeminal neuralgia (TN) by using Gamma Knife surgery (GKS). The authors compared hypesthesia and pain recurrence rates to see if statistically significant differences could be found. Methods Between July 1992 and November 2010, 737 patients presenting with TN underwent GKS and prospective evaluation at Timone University Hospital in Marseille, France. In this study the authors analyzed the cases of 497 of these patients, who participated in follow-up longer than 1 year, did not have megadolichobasilar artery- or multiple sclerosis-related TN, and underwent GKS only once; in other words, the focus was on cases of classic TN with a single radiosurgical treatment. Radiosurgery was performed with a Leksell Gamma Knife (model B, C, or Perfexion) using both MR and CT imaging targeting. A single 4-mm isocenter was positioned in the cisternal portion of the trigeminal nerve at a median distance of 7.8 mm (range 4.5-14 mm) anterior to the emergence of the nerve. A median maximum dose of 85 Gy (range 70-90 Gy) was delivered. Using empirical methods and assisted by a chart with clear cut-off periods of pain free distribution, the authors were able to divide patients who experienced freedom from pain into 3 separate groups: patients who became pain free within the first 48 hours post-GKS; those who became pain free between 48 hours and 30 days post-GKS; and those who became pain free more than 30 days after GKS. Results The median age in the 497 patients was 68.3 years (range 28.1-93.2 years). The median follow-up period was 43.75 months (range 12-174.41 months). Four hundred fifty-four patients (91.34%) were initially pain free within a median time of 10 days (range 1-459 days) after GKS. One hundred sixty-nine patients (37.2%) became pain free within the first 48 hours (Group PF(≤ 48 hours)), 194 patients (42.8%) between posttreatment Day 3 and Day 30 (Group PF((>48 hours, ≤ 30 days))), and 91 patients (20%) after 30 days post-GKS (Group PF(>30 days)). Differences in postoperative hypesthesia were found: in Group PF(≤ 48 hours) 18 patients (13.7%) developed postoperative hypesthesia, compared with 30 patients (19%) in Group PF((>48 hours, ≤ 30 days)) and 22 patients (30.6%) in Group PF(>30 days) (p = 0.014). One hundred fifty-seven patients (34.4%) who initially became free from pain experienced a recurrence of pain with a median delay of 24 months (range 0.62-150.06 months). There were no statistically significant differences between the patient groups with respect to pain recurrence: 66 patients (39%) in Group PF(≤ 48 hours) experienced pain recurrence, compared with 71 patients (36.6%) in Group PF((>48 hours, ≤ 30 days)) and 27 patients (29.7%) in Group PF(>30 days) (p = 0.515). Conclusions A substantial number of patients (169 cases, 37.2%) became pain free within the first 48 hours. The rate of hypesthesia was higher in patients who became pain free more than 30 days after GKS, with a statistically significant difference between patient groups (p = 0.014).

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system.

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

Reproductive characteristics of the predator Podisus nigrispinus fed with an insect resistant soybean variety

The objective of this research was to evaluate the effect of the insect resistant soybean genotype IAC 17 on reproductive characteristics of Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) females compared to the soybean insect susceptible genotype UFV 16. Treatments were: T1) females of P. nigrispinus fed on plants of the UFV 16 and Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) caterpillars reared on leaves of this variety; T2) females of P. nigrispinus fed on plants of the IAC 17 and A. gemmatalis caterpillars reared on leaves of this variety. Longevity of females, pre-oviposition, oviposition and pos-oviposition periods, number of eggs and egg masses/female, egg weight, interval between egg mass laying, number of eggs/egg mass, percentage of nymphs, number of nymphs/female and total number of prey killed/female of P. nigrispinus were evaluated. Most of the characteristics evaluated showed similar results between treatments, but the oviposition period was longer for females reared on the resistant genotype than on the susceptible one and the percentage of total females that laid eggs was lower on the IAC 17. Also, the resistant genotype caused higher mortality of P. nigrispinus females at the beginning of its adult stage and egg production by females of this predator was better spread along its adult stage with this resistant genotype. On the other hand, results suggest no effect of the resistant genotype on the offspring of this predator.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

Evaluation of Iowa’s 70 mph Speed Limit — 2.5 Year Update, 2009

On July 1, 2005, the State of Iowa implemented a 70 mile per hour (mph) speed limit on most rural Interstates. This document reports on a study of the safety effect of this change. Changes in speeds, traffic volume on and off the rural Interstate system (diversion), and safety (crashes) for on- and off-system roads were studied. After the change, mean and 85th percentile speeds increased by about 2 mph on rural Interstates, but speeding was reduced (the number of drivers exceeding the speed limit by 10 mph decreased from 20 per cent to about 8 per cent). Daytime and nighttime serious crashes were studied for a period of 14 and a half years prior to the change and 2 and a half years afterwards. Simple descriptive statistics reveal increases in all crash severity categories for the 2 and a half year period following the speed limit increase when compared to the most recent comparable 2 and a half year period prior to the increase. When compared to longer term trends, the increases were less pronounced in some severity levels and types, and for a few severity levels the average crash frequencies were observed to decrease. However, fatal and other serious cross-median crashes increased by relatively larger amounts as compared to expected random variation. The study also analyzed crash frequencies grouped into six-month periods, revealing similar findings.

Evaluation of Iowa's Driver Improvement Program, 2009

This study examines the effectiveness of Iowa’s Driver Improvement Program (DIP), measured as the reduction in the number of driver convictions subsequent to the DIP. The analysis involved a random sample of 9,055 drivers who had been instructed to attend DIP and corresponding data on driver convictions, crashes, and driver education training history that were provided by the Iowa Motor Vehicle Division. The sample was divided into two groups based on DIP outcome: satisfactory or unsatisfactory completion. Two evaluation periods were considered: one year after the DIP date (probation period) and the period from the 13th to 18th month after the DIP date. The evaluation of Iowa’s DIP showed that there is evidence of effectiveness in terms of reducing driver convictions subsequent to attending the DIP. Among the 6,790 (75%) drivers who completed the course satisfactorily, 73% of drivers had no actions and 93% were not involved in a crash during the probation period. Statistical tests confirmed these numbers. However, the positive effect of satisfactory completion of DIP on survival time (that is, the time until the first conviction) was not statistically significant 13 months after the DIP date. Econometric model estimation results showed that, regardless of the DIP outcome, the likelihood of conviction occurrence and frequency of subsequent convictions depends on other factors, such as age, driver history, and DIP location, and interaction effects among these factors. Low-cost, early intervention measures are suggested to enhance the effectiveness of Iowa’s DIP. These measures can include advisory and warning letters (customized based on the driver’s age) sent within the first year after the DIP date and soon after the end of the probation period, as well as a closer examination of DIP instruction across the 17 community colleges that host the program. Given the large number of suspended drivers who continued to drive, consideration should also be given to measures to reduce driving while suspended offenses.

Minimal requirements for parameters to be used for the development of predictive models for microbial source tracking. The example of the pair somatic coliphages and phages infecting Bacteroides

Several methods and approaches for measuring parameters to determine fecal sources of pollution in water have been developed in recent years. No single microbial or chemical parameter has proved sufficient to determine the source of fecal pollution. Combinations of parameters involving at least one discriminating indicator and one universal fecal indicator offer the most promising solutions for qualitative and quantitative analyses. The universal (nondiscriminating) fecal indicator provides quantitative information regarding the fecal load. The discriminating indicator contributes to the identification of a specific source. The relative values of the parameters derived from both kinds of indicators could provide information regarding the contribution to the total fecal load from each origin. It is also essential that both parameters characteristically persist in the environment for similar periods. Numerical analysis, such as inductive learning methods, could be used to select the most suitable and the lowest number of parameters to develop predictive models. These combinations of parameters provide information on factors affecting the models, such as dilution, specific types of animal source, persistence of microbial tracers, and complex mixtures from different sources. The combined use of the enumeration of somatic coliphages and the enumeration of Bacteroides-phages using different host specific strains (one from humans and another from pigs), both selected using the suggested approach, provides a feasible model for quantitative and qualitative analyses of fecal source identification.

Reproductive performance of Santa Inês ewes fed protected fat diet

The objective of this work was to evaluate the reproductive performance of Santa Inês ewes fed a diet supplemented with protected fat. Intervals from lambing to first clinical estrus and to conception, conception rate, prolificacy, live weight and body condition were determined. After lambing, 60 ewes and their offsprings were weighted and randomly assigned to three treatments, based on age, body weight and number of born lambs. Treatments consisted of: control diet, or control diet plus 30 g of protected fat, from lambing to day 25 of post-lambing (Sup25), or to day 60 of post-lambing (Sup60). Out of 60 evaluated ewes, 93.3% returned to estrus, and 74.5% got pregnant, with 73.53% lambing rate and 196.5 days lambing interval. The average periods from lambing to first estrus were 32.4, 27.2 and 35.5 days for ewes fed the control diet, Sup25, and Sup60, respectively. The intervals from lambing to conception were 45.2, 46.5 and 45.2 days, and the supplemented diets did not show differences in comparison to the control diet. Supplementation with protected fat to well-nourished Santa Inês ewes does not improve their reproductive performance.

Surgical treatment of perforated peptic ulcer. Is there a need for a change?

The aim of this study is to evaluate the risk and the results of surgical treatment for perforated peptic ulcer (PPU), to compare them through time, and to determine the current optimal surgical treatment. In a retrospective study, the charts of all the patients admitted for PPU between January 1976 and October 1991 were reviewed. The features believed to be of importance in the outcome were assessed for statistical analysis. A comparison was made between three periods of the study (1976-1980, 1981-1985, 1986-1991). 247 patients were included. Mortality was 11.7% (29/247). Factors associated with an increased mortality were: shock on admission (p = 0.01), age (p < 0.001), severe associated medical illnesses (p < 0.001) and the form of treatment (p < 0.01). After multivariate analysis, only shock on admission and associated disease remained significant. Chronic peptic ulcer disease occurred in 76% of the patients. Comparing the periods showed that age, associated illnesses, percentage of acute or subacute ulcers, mortality, as well as the number of patients, are increasing. The main determinant of surgical treatment for PPU is the patient and his/her general state. Because of the high frequency of chronic peptic ulcer disease, we believe that the gold standard in the treatment for PPU remains definitive surgery. However, in the presence of more than one risk factor, suture and patch are probably safer.

Dynamic range extension of SiPM detectors with the time-gated operation

The silicon photomultiplier (SiPM) is a novel detector technology that has undergone a fast development in the last few years, owing to its single-photon resolution and ultra-fast response time. However, the typical high dark count rates of the sensor may prevent the detection of low intensity radiation fluxes. In this article, the time-gated operation with short active periods in the nanosecond range is proposed as a solution to reduce the number of cells fired due to noise and thus increase the dynamic range. The technique is aimed at application fields that function under a trigger command, such as gated fluorescence lifetime imaging microscopy.

Forage production of elephant grass under intermittent stocking

The objective of this work was to evaluate the dry matter production of elephant grass (Pennisetum purpureum) genotypes, managed under intermittent stocking. A completely randomized design was used, with two genotypes and three replicates. The treatments consisted of factorial combinations (2x2x2) of genotypes ('BRS Kurumi' and the clone CNPGL 00‑1‑3), two light interception levels (LI) at the onset of grazing (90 and 95%), and two post‑grazing canopy heights (30 and 50 cm). A total of 24 Holstein x Zebu crossbred heifers were used. The stocking density varied in order to finish the grazing periods in two days. The interval between the defoliation, based on 95% LI, resulted in a higher leaf mass per grazing cycle. The post‑grazing height of 30 cm did not affect the number of grazing cycles but provided a greater herbage accumulation rate. The cultivar BRS Kurumi has higher pasture growth, lower rest period, and greater number of grazing cycles, which results in increased forage production in the growing season.

Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.