Rationale for the post-harvest processing of leaf mass of agricultural crops using microwave radiation

Post-harvest processing of the leaf mass of various agricultural crops has general patterns. The peculiarity of the structure of the leaves is that the amount of water contained in the leaf blade and in the midrib is approximately the same, but the area of the evaporating surface of the midrib is 10–15 times less than the area of the evaporating surface of the leaf plate. Therefore, the difference in drying modes for these parts of the leaves justifies the need for different physical methods of influencing them. The aim of the research was to substantiate experimentally the general principles of moisture removal from the leaf mass of various agricultural crops using microwave radiation. Processing in the microwave field was carried out for 1,0; 1,5; 2,0; 2,5 minutes. The treated leaf layers were dried naturally. Leaves dried by convection under natural and artificial conditions, only without microwave treatment, served as a control sample. For the post-harvest processing of plantain leaves, a combined drying method is recommended, where in the first phase the leaves are treated with microwave radiation for 2,0–2,5 minutes, depending on the thickness of the leaf layer, and then in the second phase under natural conditions for 8 hours. Microwave – processing followed by convective drying in natural conditions is considered the most compromise method for drying beet tops leaves both in terms of drying time and in terms of the energy intensity of the process. On the basis on the results of the research, the application of the most rational processes of post-harvest processing of leaf mass of agricultural crops was substantiated, which consists in their processing by microwave radiation, followed by convective drying in a natural way

Dizajniranje i vrednovanje okularnih umetaka moksifloksacin hidroklorida

The objective of the present investigation was to prepare and evaluate ocular inserts of moxifloxacin. An ocular insert was made from an aqueous dispersion of moxifloxacin, sodium alginate, polyvinyl alcohol, and dibutyl phthalate by the film casting method. The ocular insert (5.5 mm diameter) was cross-linked by CaCl2 and was coated with Eudragit S-100, RL-100, RS-100, E-100 or Eudragit L-100. The in vitro drug drainage/permeation studies were carried out using an all-glass modified Franz diffusion cell. The drug concentration and mucoadhesion time of the ocular insert were found satisfactory. Cross-linking and coating with polymers extended the drainage from inserts. The cross-linked ocular insert coated with Eudragit RL-100 showed maximum drug permeation compared to other formulations.Cilj rada bio je priprava i evaluacija okularnih umetaka moksifloksacina. Okularni umetak izrađen je od vodene suspenzije moksifloksacina, natrijevog alginata, polivinilnog alkohola i dibutil-ftalata metodom odlijevanja filma. Okularni umetak (promjera 5,5 mm) umrežen je pomoću CaCl2 i obložen Eudragitom S-100, RL-100, RS-100, E-100 ili Eudragit L-100. In vitro drenaža/permeacija lijeka proučavana je koristeći staklenu modificiranu Franzovu difuzijsku ćeliju. Koncentracija lijeka i vrijeme mukoadhezije okularnih umetaka bili su zadovoljavajući. Umrežavanje i oblaganje polimerima produljilo je drenažu iz umetaka. Umreženi okularni umetci obloženi s Eudragit RL-100 pokazali su veću permeaciju lijeka u odnosu na ostale pripravke

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3′-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5′-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5′-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5′-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3′ region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122–HCV interaction, and the broader consequences of miRNAs binding to 5′-UTR sites

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

Translation without eIF2 Promoted by Poliovirus 2A Protease

Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2Apro alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2Apro variant unable to cleave eIF4G. The level of 2Apro synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2Apro at high concentrations is necessary. The finding that 2Apro provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis

microRNA-122 stimulates translation of hepatitis C virus RNA

Hepatitis C virus (HCV) is a positive strand RNA virus that propagates primarily in the liver. We show here that the liver-specific microRNA-122 (miR-122), a member of a class of small cellular RNAs that mediate post-transcriptional gene regulation usually by repressing the translation of mRNAs through interaction with their 3′-untranslated regions (UTRs), stimulates the translation of HCV. Sequestration of miR-122 in liver cell lines strongly reduces HCV translation, whereas addition of miR-122 stimulates HCV translation in liver cell lines as well as in the non-liver HeLa cells and in rabbit reticulocyte lysate. The stimulation is conferred by direct interaction of miR-122 with two target sites in the 5′-UTR of the HCV genome. With a replication-defective NS5B polymerase mutant genome, we show that the translation stimulation is independent of viral RNA synthesis. miR-122 stimulates HCV translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage. In conclusion, the liver-specific miR-122 may contribute to HCV liver tropism at the level of translation

Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

Gene networks are an efficient route for associating candidate genes with biological processes. Here, networks are used to discover more than 15 new genes for ribosomal subunit maturation, rRNA processing, and ribosomal export from the nucleus

Xenopus Meiotic Microtubule-Associated Interactome

In metazoan oocytes the assembly of a microtubule-based spindle depends on the activity of a large number of accessory non-tubulin proteins, many of which remain unknown. In this work we isolated the microtubule-bound proteins from Xenopus eggs. Using mass spectrometry we identified 318 proteins, only 43 of which are known to bind microtubules. To integrate our results, we compiled for the first time a network of the meiotic microtubule-related interactome. The map reveals numerous interactions between spindle microtubules and the newly identified non-tubulin spindle components and highlights proteins absent from the mitotic spindle proteome. To validate newly identified spindle components, we expressed as GFP-fusions nine proteins identified by us and for first time demonstrated that Mgc68500, Loc398535, Nif3l1bp1/THOC7, LSM14A/RAP55A, TSGA14/CEP41, Mgc80361 and Mgc81475 are associated with spindles in egg extracts or in somatic cells. Furthermore, we showed that transfection of HeLa cells with siRNAs, corresponding to the human orthologue of Mgc81475 dramatically perturbs spindle formation in HeLa cells. These results show that our approach to the identification of the Xenopus microtubule-associated proteome yielded bona fide factors with a role in spindle assembly

The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation

The 3′ untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3′ UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5′ and 3′ UTRs, we found that the 3′ UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3′-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3′-ends such as the poly(A) or the 3′ UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3′ UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5′ UTR. In conclusion, we have found that the 3′-end terminal sequence can play a role in regulating the translation of CSFV

Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae