Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

High Prevalence and High Incidence of Coinfection with Hepatitis B,Hepatitis C,and Syphilis and Low Rate of Effective Vaccination against Hepatitis B in HIV-Positive Men Who Have Sex with Men with Known Date of HIV Seroconversion in Germany

Objectives

Men who have sex with men (MSM) are at higher risk for coinfection with hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis than the general population. HIV infection and these coinfections accelerate disease progression reciprocally. This study evaluated the prevalence and incidence of these coinfections in HIV1-positive MSM in Germany.

Materials and Methods

As part of a nationwide, multicenter, prospective cohort study of HIV-infected MSM, plasma samples collected yearly were screened for HBsAg and antibodies to HBc, HBs, HCV, and syphilis. Samples with indications of active HBV or HCV infection were confirmed by polymerase chain reaction. Prevalence and incidence of each infection and incidence rates per study participant were calculated, and incidences over 4-year time intervals compared.

Results

This study screened 5,445 samples from 1,843 MSM. Median age at HIV seroconversion was 33 years. Prevalences of active, cleared, and occult HBV, and of active/cleared HCV were 1.7%, 27.1%, 0.2%, and 8.2%, respectively, and 47.5% had been effectively vaccinated against HBV. Prevalence of antibodies to Treponema pallidum and of triple or quadruple sexually transmitted infections (STIs) were 39.6% and 18.9%, respectively. Prevalence of STI, cleared HBV, HBV vaccination, and history of syphilis differed significantly among age groups. Incidences of HBV, HCV, and syphilis were 2.51, 1.54, and 4.06 per 100 person-years, respectively. Incidences of HCV and syphilis increased over time. HCV incidence was significantly higher in MSM coinfected with syphilis and living in Berlin, and syphilis incidence was significantly higher for MSM living in Berlin.

Discussion

Despite extensive HBV vaccination campaigns, fewer than 50% of screened MSM were effectively vaccinated, with a high proportion of HIV-positive MSM coinfected with HBV. High rates of STI coinfections in HIV-positive MSM and increasing incidences emphasize the need for better tailored campaigns for HBV vaccination and STI prevention.     

Awareness of Sensorimotor Adaptation to Visual Rotations of Different Size

Previous studies on sensorimotor adaptation revealed no awareness of the nature of the perturbation after adaptation to an abrupt 30° rotation of visual feedback or after adaptation to gradually introduced perturbations. Whether the degree of awareness depends on the magnitude of the perturbation, though, has as yet not been tested. Instead of using questionnaires, as was often done in previous work, the present study used a process dissociation procedure to measure awareness and unawareness. A naïve, implicit group and a group of subjects using explicit strategies adapted to 20°, 40° and 60° cursor rotations in different adaptation blocks that were each followed by determination of awareness and unawareness indices. The awareness index differed between groups and increased from 20° to 60° adaptation. In contrast, there was no group difference for the unawareness index, but it also depended on the size of the rotation. Early adaptation varied between groups and correlated with awareness: The more awareness a participant had developed the more the person adapted in the beginning of the adaptation block. In addition, there was a significant group difference for savings but it did not correlate with awareness. Our findings suggest that awareness depends on perturbation size and that aware and strategic processes are differentially involved during adaptation and savings. Moreover, the use of the process dissociation procedure opens the opportunity to determine awareness and unawareness indices in future sensorimotor adaptation research.     

Response to Therapeutic Plasma Exchange as a Rescue Treatment in Clinically Isolated Syndromes and Acute Worsening of Multiple Sclerosis: A Retrospective Analysis of 90 Patients

Objectives

Experience with therapeutic plasma exchange (TPE) for acute relapses in clinically isolated syndrome (CIS) or multiple sclerosis (MS) patients has been derived from small and inhomogeneous patient populations so far. In the present study, we retrospectively evaluated features associated with TPE response in a larger cohort of CIS and MS patients with acute worsening of disease.

Participants

Ninety CIS and MS patients with acute relapses or acute worsening of symptoms were firstly treated with TPE. The population consisted of 62 women and 28 men with a median age of 38 years (range 18–69 years).

Outcome Measures

Primary endpoint was the clinical response to TPE, focused on the functional improvement of the target neurologic deficit. Secondary endpoint was an improvement in expanded disability status scale (EDSS) scoring.

Results

A clinical response to TPE was observed in 65 out of 90 patients (72.2%), with marked improvement in 18 (20.0%) and moderate improvement in 47 out of 90 patients (52.2%). The median EDSS was reduced from 3.75 before to 3.0 after TPE (p = 0.001). Response to TPE was significantly more frequent in patients with relapsing courses of disease (CIS, RR-MS, p = 0.001), no disease modifying drugs (p = 0.017), gadolinium-positive (Gd⁺) MRI lesions (p = 0.001) and EDSS ≤ 5.0 before TPE (p = 0.014). In the multiple logistic regression analysis only the detection of Gd⁺ MRI lesions was significantly altered (p = 0.004).

Conclusion

Clinical response to TPE was achieved in the majority of our patients. We identified clinical and diagnostic features in CIS and MS relapses that might be helpful to identify patients responding to TPE. Gd⁺ MRI lesions before treatment were the best predictor of the response to TPE in our cohort.     

Influence of light on ochratoxin biosynthesis by <Emphasis Type="Italic">Penicillium</Emphasis>

Light has a profound influence on ochratoxin biosynthesis by Penicillia. When incubated under constant daylight of a certain intensity, ochratoxin A biosynthesis is decreased by about 20–30% compared to incubation under constant darkness. Under day/night oscillation, the ochratoxin A polyketide synthase gene, a key gene of the ochratoxin A biosynthesis pathway, is rhythmically expressed, and moreover, the amount of ochratoxin also oscillates between the amounts produced either during constant darkness or during constant light. This indicates a partial degradation of ochratoxin A (20–30%) under light conditions until a certain lower limit is reached. This behavior is dependent on the light intensity. At 1,600 Lux, only weak effects could be observed; however, at 2,800 Lux, the effects became significant. After growth under constant light conditions, Penicillium produced ochratoxin B at amounts which are 5 times higher than after growth in constant dark or in alternating light/dark conditions. Growth experiments in the dark on medium with increasing amounts of ochratoxin A revealed that externally applied ochratoxin is moderately toxic. However, if the same growth experiments are carried out under light conditions, the growth inhibiting activity of ochratoxin A is greatly increased, indicating that light amplifies the toxic activity of ochratoxin. Because of the oscillation of the concentration of ochratoxin A during night and day incubation, Penicillium seems to have developed an adaptive mechanism to reduce the amount of ochratoxin A during daylight below a toxic level.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Morphology and physiology of the prosternal chordotonal organ of the sarcophagid fly Sarcophaga bullata (Parker)

The anatomy and the physiology of the prosternal chordotonal organ (pCO) within the prothorax of Sarcophaga bullata is analysed. Neuroanatomical studies illustrate that the approximately 35 sensory axons terminate within the median ventral association centre of the different neuromeres of the thoracico-abdominal ganglion. At the single-cell level two classes of receptor cells can be discriminated physiologically and morphologically: receptor cells with dorso-lateral branches in the mesothoracic neuromere are insensitive to frequencies below approximately 1 kHz. Receptor cells without such branches respond most sensitive at lower frequencies. Absolute thresholds vary between 0.2 and 8m/s(2) for different frequencies. The sensory information is transmitted to the brain via ascending interneurons. Functional analyses reveal a mechanical transmission of forced head rotations and of foreleg vibrations to the attachment site of the pCO. In summed action potential recordings a physiological correlate was found to stimuli with parameters of leg vibrations, rather than to those of head rotation. The data represent a first physiological study of a putative predecessor organ of an insect ear.