Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Domestic Dogs as Sentinels for West Nile Virus but not Aedes-borne Flaviviruses, Mexico.

We tested 294 domestic pet dogs in Mexico for neutralizing antibodies for mosquito-borne flaviviruses. We found high (42.6%) exposure to West Nile virus in Reynosa (northern Mexico) and low (1.2%) exposure in Tuxtla Gutierrez (southern Mexico) but very limited exposure to Aedes-borne flaviviruses. Domestic dogs may be useful sentinels for West Nile virus.

Controlled human malaria infection with Plasmodium falciparum demonstrates impact of naturally acquired immunity on virulence gene expression.

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.

Endometrial Cancer Lymphadenectomy Trial (ECLAT) (pelvic and para-aortic lymphadenectomy in patients with stage I or II endometrial cancer with high risk of recurrence; AGO-OP.6).

BACKGROUND: The impact of comprehensive pelvic and para-aortic lymphadenectomy on survival in patients with stage I or II endometrial cancer with a high risk of recurrence is not reliably documented. The side effects of this procedure, including lymphedema and lymph cysts, are evident. PRIMARY OBJECTIVE: Evaluation of the effect of comprehensive pelvic and para-aortic lymphadenectomy in the absence of bulky nodes on 5 year overall survival of patients with endometrial cancer (International Federation of Gynecology and Obstetrics (FIGO) stages I and II) and a high risk of recurrence. STUDY HYPOTHESIS: Comprehensive pelvic and para-aortic lymphadenectomy will increase 5 year overall survival from 75% (no lymphadenectomy) to 83%, corresponding to a hazard ratio of 0.65. TRIAL DESIGN: Open label, randomized, controlled trial. In arm A, a total hysterectomy plus bilateral salpingo-oophorectomy is performed. In arm B, in addition, a systematic pelvic and para-aortic lymphadenectomy up to the level of the left renal vein is performed. For all patients, vaginal brachytherapy and adjuvant chemotherapy (carboplatin/paclitaxel) are recommended. MAJOR INCLUSION CRITERIA: Patients with histologically confirmed endometrial cancer stages pT1b-pT2, all histological subtypes, and pT1a endometrioid G3, serous, clear cell, or carcinosarcomas can be included when bulky nodes are absent. When hysterectomy has already been performed (eg, for presumed low risk endometrial cancer), study participation is also possible. EXCLUSION CRITERIA: Patients with pT1a, G1 or 2 of type 1 histology or uterine sarcomas (except for carcinosarcomas), endometrial cancers of FIGO stage III or IV (except for microscopic lymph node metastases) or visual extrauterine disease. PRIMARY ENDPOINT: Overall survival calculated from the date of randomization until death. SAMPLE SIZE: 640 patients will be enrolled in the study. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: At present, 252 patients have been recruited. Based on this, accrual should be completed in 2025. Results should be presented in 2031. TRIAL REGISTRATION: NCT03438474.

Identification of a conserved var gene in different Plasmodium falciparum strains.

BACKGROUND: The multicopy var gene family of Plasmodium falciparum is of crucial importance for pathogenesis and antigenic variation. So far only var2csa, the var gene responsible for placental malaria, was found to be highly conserved among all P. falciparum strains. Here, a new conserved 3D7 var gene (PF3D7_0617400) is identified in several field isolates. METHODS: DNA sequencing, transcriptional analysis, Cluster of Differentiation (CD) 36-receptor binding, indirect immunofluorescence with PF3D7_0617400-antibodies and quantification of surface reactivity against semi-immune sera were used to characterize an NF54 clone and a Gabonese field isolate clone (MOA C3) transcribing the gene. A population of 714 whole genome sequenced parasites was analysed to characterize the conservation of the locus in African and Asian isolates. The genetic diversity of two var2csa fragments was compared with the genetic diversity of 57 microsatellites fragments in field isolates. RESULTS: PFGA01_060022400 was identified in a Gabonese parasite isolate (MOA) from a chronic infection and found to be 99% identical with PF3D7_0617400 of the 3D7 genome strain. Transcriptional analysis and immunofluorescence showed expression of the gene in an NF54 and a MOA clone but CD36 binding assays and surface reactivity to semi-immune sera differed markedly in the two clones. Long-read Pacific bioscience whole genome sequencing showed that PFGA01_060022400 is located in the internal cluster of chromosome 6. The full length PFGA01_060022400 was detected in 36 of 714 P. falciparum isolates and 500 bp fragments were identified in more than 100 isolates. var2csa was in parts highly conserved (He = 0) but in other parts as variable (He = 0.86) as the 57 microsatellites markers (He = 0.8). CONCLUSIONS: Individual var gene sequences exhibit conservation in the global parasite population suggesting that purifying selection may limit overall genetic diversity of some var genes. Notably, field and laboratory isolates expressing the same var gene exhibit markedly different phenotypes.

Cell fusing agent virus (Flavivirus) infection in Aedes aegypti in Texas: seasonality, comparison by trap type, and individual viral loads.

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.

A supermassive black hole in an ultra-compact dwarf galaxy.

Ultra-compact dwarf galaxies are among the densest stellar systems in the Universe. These systems have masses of up to 2 × 10(8) solar masses, but half-light radii of just 3-50 parsecs. Dynamical mass estimates show that many such dwarfs are more massive than expected from their luminosity. It remains unclear whether these high dynamical mass estimates arise because of the presence of supermassive black holes or result from a non-standard stellar initial mass function that causes the average stellar mass to be higher than expected. Here we report adaptive optics kinematic data of the ultra-compact dwarf galaxy M60-UCD1 that show a central velocity dispersion peak exceeding 100 kilometres per second and modest rotation. Dynamical modelling of these data reveals the presence of a supermassive black hole with a mass of 2.1 × 10(7) solar masses. This is 15 per cent of the object's total mass. The high black hole mass and mass fraction suggest that M60-UCD1 is the stripped nucleus of a galaxy. Our analysis also shows that M60-UCD1's stellar mass is consistent with its luminosity, implying a large population of previously unrecognized supermassive black holes in other ultra-compact dwarf galaxies.

A pilot study on the influence of mouth configuration and torso on singing voice directivity.

Directivity of speech and singing is determined primarily by the morphology of a person, i.e., head size, torso dimensions, posture, and vocal tract. Previous works have suggested from measurements that voice directivity in singing is controlled unintentionally by spectral emphasis in the range of 2-4 kHz. The attempt is made to try to identify to what extent voice directivity is affected by the mouth configuration and the torso. Therefore, simulations, together with measurements that investigate voice directivity in more detail, are presented. Simulations are presented for a piston in an infinite baffle, a radiating spherical cap, and an extended spherical cap model, taking into account transverse propagation modes. Measurements of a classical singer, an amateur singer, and a head and torso simulator are undertaken simultaneously in the horizontal and vertical planes. In order to assess differences of voice directivity common metrics, e.g., horizontal and vertical directivity indexes, are discussed and compared to improved alternatives. The measurements and simulations reveal that voice directivity in singing is affected if the mouth opening is changed significantly. The measurements show that the torso generates side lobes due to diffraction and reflections at frequencies related to the torso's dimensions.

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance.

BACKGROUND: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. RESULTS: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0-3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. CONCLUSIONS: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates.

What Are the Reference Values and Associated Factors for Center-edge Angle and Alpha Angle? A Population-based Study.

BACKGROUND: The available evidence regarding normal ranges for the center-edge angle and the alpha angle derives from a few small studies, and associated factors such as sex and anthropometric factors have not been well evaluated. Knowing more about normal values for these parameters is critical, because this can inform decisions about when to perform elective hip preservation surgery. Population-level studies would provide considerable clarity on these issues, but to our knowledge, no such studies are available. QUESTIONS/PURPOSES: The purposes of this study were (1) to use MRI in patients of a population-based study to establish normal values for the alpha and center-edge angles in the normal adult hip; and (2) to determine whether age, sex, or anthropometric variables were associated with differences in these values. METHODS: We used MRI images (1.5 T) of 3226 participants of the Study of Health in Pomerania (SHIP). SHIP is a population-based study that started with 4308 participants in 1997. Participants were recruited randomly from official inhabitant data files as a stratified cluster sample of the population from a defined region in northeastern Germany. To ensure a representative epidemiologic cohort, stratification variables were sex, age, and city of residence. Between 2008 and 2012, 1094 of these participants underwent whole-body MRI with pelvic sequences in the second followup. In parallel, the next cohort, SHIP-Trend, started with the same protocol in which 2132 participants underwent MRI examination. Reference values were calculated by quantile regressions for the 2.5 and 97.5 percentiles. Associations with the demographic features sex, age, weight, height, body mass index (BMI), and waist circumference were analyzed by bivariate linear regression models. RESULTS: The mean center-edge angle was 31° (± 7°) with a corresponding calculated normal range of 17° to 45°. The mean alpha angle was 55° (± 8°) with a corresponding calculated normal range between 39° and 71°. Men (30° ± 7°) had a lower center-edge angle than women (32° ± 8°) (p < 0.001, ß = 1.4°; 95% confidence interval [CI], 0.9°-1.9°) and a higher alpha angle (57° ± 8° versus 52° ± 7°, p < 0.001, ß = 5.7°; 95% CI, 5.2°-6.3°). Moreover, a higher center-edge angle for the left side was found (33° ± 8° versus 30° ± 8°, p < 0.001, ß =3.2°; 95% CI, 3.0°-3.4°). In addition to sex, we found that age, height, waist circumference, and BMI affected both center-edge angle and alpha angle. Weight was associated with the alpha angle as well. Because of these associations, age- and sex-adjusted reference values with belonging formulas were calculated. CONCLUSIONS: The range of normal center-edge and alpha angles is quite wide. Therefore, only markedly abnormal angles may be associated with pathology. Moreover, center-edge angle and alpha angle are associated with age, sex, and anthropometric factors, which have to be taken into account for better interpretation. CLINICAL RELEVANCE: The association of abnormal radiographic values with true clinical hip pathology is tenuous at best. Assuming that a patient with an abnormal radiograph requires treatment is unwise. The clinical picture has to be substantial for therapeutic decisions.

New Mycobacterium tuberculosis Complex Sublineage, Brazzaville, Congo.

Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC.

Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser.

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

Energy-dependent expansion of .177 caliber hollow-point air gun projectiles.

Amongst hundreds of different projectiles for air guns available on the market, hollow-point air gun pellets are of special interest. These pellets are characterized by a tip or a hollowed-out shape in their tip which, when fired, makes the projectiles expand to an increased diameter upon entering the target medium. This results in an increase in release of energy which, in turn, has the potential to cause more serious injuries than non-hollow-point projectiles. To the best of the authors' knowledge, reliable data on the terminal ballistic features of hollow-point air gun projectiles compared to standard diabolo pellets have not yet been published in the forensic literature. The terminal ballistic performance (energy-dependent expansion and penetration) of four different types of .177 caliber hollow-point pellets discharged at kinetic energy levels from approximately 3 J up to 30 J into water, ordnance gelatin, and ordnance gelatin covered with natural chamois as a skin simulant was the subject of this investigation. Energy-dependent expansion of the tested hollow-point pellets was observed after being shot into all investigated target media. While some hollow-point pellets require a minimum kinetic energy of approximately 10 J for sufficient expansion, there are also hollow-point pellets which expand at kinetic energy levels of less than 5 J. The ratio of expansion (RE, calculated by the cross-sectional area (A) after impact divided by the cross-sectional area (A 0) of the undeformed pellet) of hollow-point air gun pellets reached values up of to 2.2. The extent of expansion relates to the kinetic energy of the projectile with a peak for pellet expansion at the 15 to 20 J range. To conclude, this work demonstrates that the hollow-point principle, i.e., the design-related enlargement of the projectiles' frontal area upon impact into a medium, does work in air guns as claimed by the manufacturers.

Penetrating chest trauma caused by a blank cartridge actuated rubber ball projectile: case presentation and ballistic investigation of an uncommon weapon type.

Recently, an increasing number of an uncommon weapon type based on a caliber 6-mm Flobert blank cartridge actuated revolver which discharges 10-mm-diameter rubber ball projectiles has been confiscated by police authorities following criminal offenses. A recent trauma case presenting with a penetrating chest injury occasioned an investigation into the basic ballistic parameters of this type of weapon. Kinetic energy E of the test projectiles was calculated between 5.8 and 12.5 J. Energy density ED of the test projectiles was close to or higher than the threshold energy density of human skin. It can be concluded that penetrating skin injuries due to free-flying rubber ball projectiles discharged at close range cannot be ruled out. However, in case of a contact shot, the main injury potential of this weapon type must be attributed to the high energy density of the muzzle gas jet which may, similar to well-known gas or alarm weapons, cause life-threatening or even lethal injuries.

Single mimivirus particles intercepted and imaged with an X-ray laser.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Sporozoite Route of Infection Influences In Vitro var Gene Transcription of Plasmodium falciparum Parasites From Controlled Human Infections.

BACKGROUND: Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. METHODS: Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. RESULTS: At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P = .03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa CONCLUSIONS: The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.

Influence of pellet seating on the external ballistic parameters of spring-piston air guns.

In firearm examiners' and forensic specialists' casework as well as in air gun proof testing, reliable measurement of the weapon's muzzle velocity is indispensable. While there are standardized and generally accepted procedures for testing the performance of air guns, the method of seating the diabolo pellets deeper into the breech of break barrel spring-piston air guns has not found its way into standardized test procedures. The influence of pellet seating on the external ballistic parameters was investigated using ten different break barrel spring-piston air guns. Test shots were performed with the diabolo pellets seated 2 mm deeper into the breech using a pellet seater. The results were then compared to reference shots with conventionally loaded diabolo pellets. Projectile velocity was measured with a high-precision redundant ballistic speed measurement system. In eight out of ten weapons, the muzzle energy increased significantly when the pellet seater was used. The average increase in kinetic energy was 31 % (range 9-96 %). To conclude, seating the pellet even slightly deeper into the breech of spring-piston air guns might significantly alter the muzzle energy. Therefore, it is strongly recommended that this effect is taken into account when accurate and reliable measurements of air gun muzzle velocity are necessary.

Modeling internal ballistics of gas combustion guns.

Potato guns are popular homemade guns which work on the principle of gas combustion. They are usually constructed for recreational rather than criminal purposes. Yet some serious injuries and fatalities due to these guns are reported. As information on the internal ballistics of homemade gas combustion-powered guns is scarce, it is the aim of this work to provide an experimental model of the internal ballistics of these devices and to investigate their basic physical parameters. A gas combustion gun was constructed with a steel tube as the main component. Gas/air mixtures of acetylene, hydrogen, and ethylene were used as propellants for discharging a 46-mm caliber test projectile. Gas pressure in the combustion chamber was captured with a piezoelectric pressure sensor. Projectile velocity was measured with a ballistic speed measurement system. The maximum gas pressure, the maximum rate of pressure rise, the time parameters of the pressure curve, and the velocity and path of the projectile through the barrel as a function of time were determined according to the pressure-time curve. The maximum gas pressure was measured to be between 1.4 bar (ethylene) and 4.5 bar (acetylene). The highest maximum rate of pressure rise was determined for hydrogen at (dp/dt)max = 607 bar/s. The muzzle energy was calculated to be between 67 J (ethylene) and 204 J (acetylene). To conclude, this work provides basic information on the internal ballistics of homemade gas combustion guns. The risk of injury to the operator or bystanders is high, because accidental explosions of the gun due to the high-pressure rise during combustion of the gas/air mixture may occur.