Comparison of soil microbial communities from two distinct karst areas in Hungary

Karst areas belong to the most exposed terrestrial ecosystems, therefore their study have a priority task in Hungary, as well. The aim of this study was to compare the structure, activity and diversity of soil microbial communities from two distinct Hungarian karst areas (Aggtelek NP and Tapolca-basin). Soil samples were taken three times from 6 distinct sites, from different depths. Soil microbial biomass C (MBC), microbial biomass N (MBN), basal respiration (BRESP) and substrate induced respiration (SIR) were measured. The phylogenetic diversity of bacterial communities was compared by Denaturing Gradient Gel Electrophoresis (DGGE). The highest MBC, MBN, BRESP and SIR values were measured in the rendzina soil from Aggtelek. On the basis of biomass and respiration measurements, microbial communities differentiated mainly according to soil depths whereas DGGE profiles of bacterial communities resulted in groups mainly according to sampling sites.     

An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (kcat) of the enzyme complex increased 2- to 3-fold whereas the Km for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.     

Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.     

Microbial transformation of neutral fraction and upgraded neutral fraction of Polish tall oil

Summary Microbial transformations of neutral fraction (NF) and upgraded neutral fraction (UNF) of Polish tall oil byMycobacterium sp. MB 3683 were performed. Final metabolites and yields were compared to bioconversion of pure -sitosterol. Additionally, origin of a new metabolite —5-androsta-3,6,17-trione was proved by transformation of UNF in the presence of labeled -sitosterol.     

Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.     

Changes in the Frequency Structure of a Mating Call Decrease Its Attractiveness to Females in the Cricket Frog Acris crepitans blanchardi

In many species, females often prefer male signals that are more complex than in nature or beyond the range of calls naturally produced by conspecific males in spectral, temporal and amplitude features. In this study we examined both the ability of females to recognize signals outside the normal range of spectral frequency variation seen in male advertisement calls, and the influence of increasing call complexity by adding spectral components to enhance the attractiveness of a male advertisement call in the cricket frog Acris crepitans blanchardi, while keeping its amplitude constant. We used two different natural male call groups and created the following synthetic call groups: with a dominant frequency at 3500 Hz, i.e. at the normal dominant frequency with a frequency band within the sensitivity range of the inner ear basilar papilla; with a dominant frequency at 700 Hz, i.e. outside the normal range of variation and with a frequency band outside the sensitivity range of the basilar papilla but within the range of the amphibian papilla; with two dominant frequencies, one at 700 Hz and another at 3500 Hz, stimulating the basilar and amphibian papilla simultaneously. In double choice experiments we tested all combinations of the three call groups, and we tested the 3500 Hz call groups against the same natural call groups. Additionally, we tested the 700 Hz call groups against white noise to see whether these signals are meaningful in mate choice. Females preferred 3500 Hz call groups over all other call groups. The synthetic call group was as attractive to females as the same natural call group. The 700 Hz call group was not meaningful in mate choice. The combined (700 Hz + 3500 Hz) call group was significantly less attractive to females than the 3500 Hz call group. Thus, making a call more spectrally complex without increasing its overall amplitude decreases its attractiveness to cricket frog females.     

Mortality,Morbidity and Health-Seeking Behaviour during the Ebola Epidemic 2014–2015 in Monrovia Results from a Mobile Phone Survey

Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0.33/10,000 persons/day (95%CI:0.25–0.43) and an Ebola-specific mortality rate of 0.06/10,000 persons/day (95%-CI:0.03–0.11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.     

Non-Attended Representations are Perceptual Rather than Unconscious in Nature

Introspectively we experience a phenomenally rich world. In stark contrast, many studies show that we can only report on the few items that we happen to attend to. So what happens to the unattended objects? Are these consciously processed as our first person perspective would have us believe, or are they – in fact – entirely unconscious? Here, we attempt to resolve this question by investigating the perceptual characteristics of visual sensory memory. Sensory memory is a fleeting, high-capacity form of memory that precedes attentional selection and working memory. We found that memory capacity benefits from figural information induced by the Kanizsa illusion. Importantly, this benefit was larger for sensory memory than for working memory and depended critically on the illusion, not on the stimulus configuration. This shows that pre-attentive sensory memory contains representations that have a genuinely perceptual nature, suggesting that non-attended representations are phenomenally experienced rather than unconscious.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.