A global analysis of the impact of COVID-19 stay-at-home restrictions on crime.

The stay-at-home restrictions to control the spread of COVID-19 led to unparalleled sudden change in daily life, but it is unclear how they affected urban crime globally. We collected data on daily counts of crime in 27 cities across 23 countries in the Americas, Europe, the Middle East and Asia. We conducted interrupted time series analyses to assess the impact of stay-at-home restrictions on different types of crime in each city. Our findings show that the stay-at-home policies were associated with a considerable drop in urban crime, but with substantial variation across cities and types of crime. Meta-regression results showed that more stringent restrictions over movement in public space were predictive of larger declines in crime.

The hyperthermophilic partners Nanoarchaeum and Ignicoccus stabilize their tRNA T-loops via different but structurally equivalent modifications.

The universal L-shaped tertiary structure of tRNAs is maintained with the help of nucleotide modifications within the D- and T-loops, and these modifications are most extensive within hyperthermophilic species. The obligate-commensal Nanoarchaeum equitans and its phylogenetically-distinct host Ignicoccus hospitalis grow physically coupled under identical hyperthermic conditions. We report here two fundamentally different routes by which these archaea modify the key conserved nucleotide U54 within their tRNA T-loops. In N. equitans, this nucleotide is methylated by the S-adenosylmethionine-dependent enzyme NEQ053 to form m5U54, and a recombinant version of this enzyme maintains specificity for U54 in Escherichia coli. In N. equitans, m5U54 is subsequently thiolated to form m5s2U54. In contrast, I. hospitalis isomerizes U54 to pseudouridine prior to methylating its N1-position and thiolating the O4-position of the nucleobase to form the previously uncharacterized nucleotide m1s4Ψ. The methyl and thiol groups in m1s4Ψ and m5s2U are presented within the T-loop in a spatially identical manner that stabilizes the 3'-endo-anti conformation of nucleotide-54, facilitating stacking onto adjacent nucleotides and reverse-Hoogsteen pairing with nucleotide m1A58. Thus, two distinct structurally-equivalent solutions have evolved independently and convergently to maintain the tertiary fold of tRNAs under extreme hyperthermic conditions.

Reductive Evolution and Diversification of C5-Uracil Methylation in the Nucleic Acids of Mollicutes.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U-modifying enzymes that is highly dynamic and has undergone horizontal transfer.

Helcococcus kunzii methyltransferase Erm(47) responsible for MLSB resistance is induced by diverse ribosome-targeting antibiotics.

OBJECTIVES: To determine the mechanism of induction of erm(47) and its atypical expression in the Gram-positive opportunistic pathogen Helcococcus kunzii, where it confers resistance to a subset of clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics. METHODS: The resistant H. kunzii clinical isolate UCN99 was challenged with subinhibitory concentrations of a wide range of ribosome-targeting drugs. The methylation status of the H. kunzii ribosomal RNA at the MLSB binding site was then determined using an MS approach and was correlated with any increase in resistance to the drugs. RESULTS: The H. kunzii erm(47) gene encodes a monomethyltransferase. Expression is induced by subinhibitory concentrations of the macrolide erythromycin, as is common for many erm genes, and surprisingly also by 16-membered macrolide, lincosamide, streptogramin, ketolide, chloramphenicol and linezolid antibiotics, all of which target the 50S ribosomal subunit. No induction was detected with spectinomycin, which targets the 30S subunit. CONCLUSIONS: The structure of the erm(47) leader sequence functions as a hair trigger for the induction mechanism that expresses resistance. Consequently, translation of the erm(47) mRNA is tripped by MLSB compounds and also by drugs that target the 50S ribosomal subunit outside the MLSB site. Expression of erm(47) thus extends previous assumptions about how erm genes can be induced.

Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR.

Strains of the Pasteurellaceae bacteria Pasteurella multocida and Mannheimia haemolytica are major etiological agents of bovine respiratory disease (BRD). Treatment of BRD with antimicrobials is becoming more challenging due to the increasing occurrence of resistance in infecting strains. In Pasteurellaceae strains exhibiting resistance to multiple antimicrobials including aminoglycosides, beta-lactams, macrolides and sulfonamides, the resistance determinants are often chromosomally encoded within integrative and conjugative elements (ICEs). To gain a more comprehensive picture of ICE structures, we sequenced the genomes of six strains of P. multocida and four strains of M. haemolytica; all strains were independent isolates and eight of them were multiple-resistant. ICE sequences varied in size from 49 to 79 kb, and were comprised of an array of conserved genes within a core region and varieties of resistance genes within accessory regions. These latter regions mainly account for the variation in the overall ICE sizes. From the sequence data, we developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures. Application of this assay on 75 isolates of P. multocida and M. haemolytica reveals how the presence and structures of ICEs are related to their antibiotic resistance phenotypes. The assay is also applicable to other members of the Pasteurellaceae family including Histophilus somni and indicates how clustering and dissemination of the resistance genes came about.

Bypassing rRNA methylation by RsmA/Dim1during ribosome maturation in the hyperthermophilic archaeon Nanoarchaeum equitans.

In all free-living organisms a late-stage checkpoint in the biogenesis of the small ribosomal subunit involves rRNA modification by an RsmA/Dim1 methyltransferase. The hyperthermophilic archaeon Nanoarchaeum equitans, whose existence is confined to the surface of a second archaeon, Ignicoccus hospitalis, lacks an RsmA/Dim1 homolog. We demonstrate here that the I. hospitalis host possesses the homolog Igni_1059, which dimethylates the N6-positions of two invariant adenosines within helix 45 of 16S rRNA in a manner identical to other RsmA/Dim1 enzymes. However, Igni_1059 is not transferred from I. hospitalis to N. equitans across their fused cell membrane structures and the corresponding nucleotides in N. equitans 16S rRNA remain unmethylated. An alternative mechanism for ribosomal subunit maturation in N. equitans is suggested by sRNA interactions that span the redundant RsmA/Dim1 site to introduce 2΄-O-ribose methylations within helices 44 and 45 of the rRNA.

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni.

Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2'-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2'-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2'-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2'-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.

Open source approaches to establishing Roseobacter clade bacteria as synthetic biology chassis for biogeoengineering.

Aim. The nascent field of bio-geoengineering stands to benefit from synthetic biologists' efforts to standardise, and in so doing democratise, biomolecular research methods. Roseobacter clade bacteria comprise 15-20% of oceanic bacterio-plankton communities, making them a prime candidate for establishment of synthetic biology chassis for bio-geoengineering activities such as bioremediation of oceanic waste plastic. Developments such as the increasing affordability of DNA synthesis and laboratory automation continue to foster the establishment of a global 'do-it-yourself' research community alongside the more traditional arenas of academe and industry. As a collaborative group of citizen, student and professional scientists we sought to test the following hypotheses: (i) that an incubator capable of cultivating bacterial cells can be constructed entirely from non-laboratory items, (ii) that marine bacteria from the Roseobacter clade can be established as a genetically tractable synthetic biology chassis using plasmids conforming to the BioBrick(TM) standard and finally, (iii) that identifying and subcloning genes from a Roseobacter clade species can readily by achieved by citizen scientists using open source cloning and bioinformatic tools. Method. We cultivated three Roseobacter species, Roseobacter denitrificans, Oceanobulbus indolifexand Dinoroseobacter shibae. For each species we measured chloramphenicol sensitivity, viability over 11 weeks of glycerol-based cryopreservation and tested the effectiveness of a series of electroporation and heat shock protocols for transformation using a variety of plasmid types. We also attempted construction of an incubator-shaker device using only publicly available components. Finally, a subgroup comprising citizen scientists designed and attempted a procedure for isolating the cold resistance anf1 gene from Oceanobulbus indolifexcells and subcloning it into a BioBrick(TM) formatted plasmid. Results. All species were stable over 11 weeks of glycerol cryopreservation, sensitive to 17 µg/mL chloramphenicol and resistant to transformation using the conditions and plasmids tested. An incubator-shaker device, 'UCLHack-12' was assembled and used to cultivate sufficient quantity of Oceanobulbus indolifexcells to enable isolation of the anf1 gene and its subcloning into a plasmid to generate the BioBrick(TM) BBa_K729016. Conclusion.The process of 'de-skilling' biomolecular techniques, particularly for relatively under-investigated organisms, is still on-going. However, our successful cell growth and DNA manipulation experiments serve to indicate the types of capabilities that are now available to citizen scientists. Science democratised in this way can make a positive contribution to the debate around the use of bio-geoengineering to address oceanic pollution or climate change.

Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing.

Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease.

Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides.

Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA.

Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m5U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

Discrimination between benign, primary and secondary malignancies in lymph nodes from the head and neck utilising Raman spectroscopy and multivariate analysis.

BACKGROUND: The potential use of Raman spectroscopy (RS) for the detection of malignancy within lymph nodes of the head and neck was evaluated. RS measures the presence of biomolecules by the inelastic scattering of light within cells and tissues. This can be performed in vivo in real-time. METHODS: 103 lymph nodes were collected from 23 patients undergoing surgery for suspicious lymph nodes. Five pathologies, defined by consensus histopathology, were collected including reactive nodes (benign), Hodgkin's and non-Hodgkin's lymphomas, metastases from both squamous cell carcinomas and adenocarcinomas. Raman spectra were measured with 830 nm excitation from numerous positions on each biopsy. Spectral diagnostic models were constructed using principal component analysis followed by linear discriminant analysis (PCA-LDA), and by partial least squares discriminant analysis (PLS-DA) for comparison. Two-group models were constructed to distinguish between reactive and malignant nodes, and three-group models to distinguish between the benign, primary and secondary conditions. RESULTS: Results were validated using a repeated subsampling procedure. Sensitivities and specificities of 90% and 86% were obtained using PCA-LDA, and 89% and 88% using PLS-DA, for the two-group models. Both PCA-LDA and PLS-DA models were also found to be very successful at discriminating between pathologies in the three-group models achieving sensitivities and specificities of over 78% and 89% for PCA-LDA, and over 81% and 89% for PLS-DA for all three pathology groups. CONCLUSION: Raman spectroscopy and chemometric techniques can be successfully utilised in combination for discriminating between different cancerous conditions of lymph nodes from the head and neck.

Multiplex PCR to identify macrolide resistance determinants in Mannheimia haemolytica and Pasteurella multocida.

The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLS(B) (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.

A single methyltransferase YefA (RlmCD) catalyses both m5U747 and m5U1939 modifications in Bacillus subtilis 23S rRNA.

Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m(5)U modifications. However, as previously shown, the m(5)U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m(5)U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m(5)U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.

Combinations of macrolide resistance determinants in field isolates of Mannheimia haemolytica and Pasteurella multocida.

Respiratory tract infections in cattle are commonly associated with the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. These infections can generally be successfully treated in the field with one of several groups of antibiotics, including macrolides. A few recent isolates of these species exhibit resistance to veterinary macrolides with phenotypes that fall into three distinct classes. The first class has type I macrolide, lincosamide, and streptogramin B antibiotic resistance and, consistent with this, the 23S rRNA nucleotide A2058 is monomethylated by the enzyme product of the erm(42) gene. The second class shows no lincosamide resistance and lacks erm(42) and concomitant 23S rRNA methylation. Sequencing of the genome of a representative strain from this class, P. multocida 3361, revealed macrolide efflux and phosphotransferase genes [respectively termed msr(E) and mph(E)] that are arranged in tandem and presumably expressed from the same promoter. The third class exhibits the most marked drug phenotype, with high resistance to all of the macrolides tested, and possesses all three resistance determinants. The combinations of erm(42), msr(E), and mph(E) are chromosomally encoded and intermingled with other exogenous genes, many of which appear to have been transferred from other members of the Pasteurellaceae. The presence of some of the exogenous genes explains recent reports of resistance to additional drug classes. We have expressed recombinant versions of the erm(42), msr(E), and mph(E) genes within an isogenic Escherichia coli background to assess their individually contributions to resistance. Our findings indicate what types of compounds might have driven the selection for these resistance determinants.

A novel Erm monomethyltransferase in antibiotic-resistant isolates of Mannheimia haemolytica and Pasteurella multocida.

Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.

Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases.

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m(5)U54 in tRNAs, RlmC (formerly RumB) that modifies m(5)U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m(5)U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m(5)U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m(5)U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m(5)U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.

The influence of the extensional viscosity of very low concentrations of high molecular mass water-soluble polymers on atomisation and droplet impact.

BACKGROUND: Water-soluble polymers are increasingly added to herbicide and pesticide formulations at very low concentrations (100-1000 mg L(-1)) in order to control the spray characteristics, notably to reduce spray drift and influence droplet bounce. The incorporation of polymeric adjuvants improves the efficacy of the spray solutions, thus enabling crop growers to maximise the performance of agrochemical sprays at lower dose rates of active ingredient. It is important to establish a fundamental understanding of how polymers influence the processes involved in droplet deposition. RESULTS: The shear and extensional viscosities of a series of high molecular mass (M(w)) poly(acrylamides) (M(w) approximately 10(6)-10(7)) have been determined at very low concentrations (100-1000 mg L(-1)). The polymer solutions demonstrated typical shear thinning characteristics under shear, and strain hardening behaviour under extension above a critical strain rate. The presence of the polymers was shown to increase the size of droplets produced in atomisation using an agricultural spray nozzle, as measured by laser diffraction. This was attributed to the increase in the extensional viscosity at the strain rates generated under pressure in the spray nozzle and was a function of both polymer concentration and M(w). In addition, the presence of polymer was found to have a significant influence on droplet bounce. CONCLUSIONS: The presence of very low concentrations of high molecular mass poly(acrylamides) significantly influences the size of droplets formed on atomisation and subsequent bounce characteristics. Large extensional viscosities generated above a critical strain rate are responsible for both processes.

Caenorhabditis elegans intersectin: a synaptic protein regulating neurotransmission.

Intersectin is a multifunctional protein that interacts with components of the endocytic and exocytic pathways, and it is also involved in the control of actin dynamics. Drosophila intersectin is required for viability, synaptic development, and synaptic vesicle recycling. Here, we report the characterization of intersectin function in Caenorhabditis elegans. Nematode intersectin (ITSN-1) is expressed in the nervous system, and it is enriched in presynaptic regions. The C. elegans intersectin gene (itsn-1) is nonessential for viability. In addition, itsn-1-null worms do not display any evident phenotype, under physiological conditions. However, they display aldicarb-hypersensitivity, compatible with a negative regulatory role of ITSN-1 on neurotransmission. ITSN-1 physically interacts with dynamin and EHS-1, two proteins involved in synaptic vesicle recycling. We have previously shown that EHS-1 is a positive modulator of synaptic vesicle recycling in the nematode, likely through modulation of dynamin or dynamin-controlled pathways. Here, we show that ITSN-1 and EHS-1 have opposite effects on aldicarb sensitivity, and on dynamin-dependent phenotypes. Thus, the sum of our results identifies dynamin, or a dynamin-controlled pathway, as a potential target for the negative regulatory role of ITSN-1.

UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development.

The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.