Close Sequence Similarity Research Articles

Roles of the MYB94/FUSED LEAVES1 (ZmFDL1) and GLOSSY2 (ZmGL2) genes in cuticle biosynthesis and potential impacts on Fusarium verticillioides growth on maize silks.

Maize silks, the stigmatic portions of the female flowers, have an important role in reproductive development. Silks also provide entry points for pathogens into host tissues since fungal hyphae move along the surface of the silks to reach the site of infection, i.e., the developing kernel. The outer extracellular surface of the silk is covered by a protective hydrophobic cuticle, comprised of a complex array of long-chain hydrocarbons and small amounts of very long chain fatty acids and fatty alcohols. This work illustrates that two previously characterized cuticle-related genes separately exert roles on maize silk cuticle deposition and function. ZmMYB94/FUSED LEAVES 1 (ZmFDL1) MYB transcription factor is a key regulator of cuticle deposition in maize seedlings. The ZmGLOSSY2 (ZmGL2) gene, a putative member of the BAHD superfamily of acyltransferases with close sequence similarity to the Arabidopsis AtCER2 gene, is involved in the elongation of the fatty acid chains that serve as precursors of the waxes on young leaves. In silks, lack of ZmFDL1 action generates a decrease in the accumulation of a wide number of compounds, including alkanes and alkenes of 20 carbons or greater and affects the expression of cuticle-related genes. These results suggest that ZmFDL1 retains a regulatory role in silks, which might be exerted across the entire wax biosynthesis pathway. Separately, a comparison between gl2-ref and wild-type silks reveals differences in the abundance of specific cuticular wax constituents, particularly those of longer unsaturated carbon chain lengths. The inferred role of ZmGL2 is to control the chain lengths of unsaturated hydrocarbons. The treatment of maize silks with Fusarium verticillioides conidia suspension results in altered transcript levels of ZmFDL1 and ZmGL2 genes. In addition, an increase in fungal growth was observed on gl2-ref mutant silks 72 hours after Fusarium infection. These findings suggest that the silk cuticle plays an active role in the response to F. verticillioides infection.

Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

Mutations conferring SO42− pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant

Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42−, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42− pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42− transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42− pump activity. Notably, the MrHR and SyHR mutants did not show SO42− induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42− in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42− into their centers after illumination and release it before the ends of the photoreactions.

Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

Paleobiogeographical origins of Fasciola hepatica and F. gigantica in light of new DNA sequence characteristics of F. nyanzae from hippopotamus.

Fascioliasis is a highly pathogenic disease affecting humans and livestock worldwide. It is caused by the liver flukes Fasciola hepatica transmitted by Galba/Fossaria lymnaeid snails in Europe, Asia, Africa, the Americas and Oceania, and F. gigantica transmitted by Radix lymnaeids in Africa and Asia. An evident founder effect appears in genetic studies as the consequence of their spread by human-guided movements of domestic ruminants, equines and Old World camelids in the post-domestication period from the beginning of the Neolithic. Establishing the geographical origins of fasciolid expansion is multidisciplinary crucial for disease assessment. Sequencing of selected nuclear ribosomal and mitochondrial DNA markers of F. nyanzae infecting hippopotamuses (Hippopotamus amphibius) in South Africa and their comparative analyses with F. hepatica and F. gigantica, and the two Fascioloides species, Fs. jacksoni from Asian elephants and Fs. magna from Holarctic cervids, allow to draw a tuned-up evolutionary scenario during the pre-domestication period. Close sequence similarities indicate a direct derivation of F. hepatica and F. gigantica from F. nyanzae by speciation after host capture phenomena. Phylogenetic reconstruction, genetic distances and divergence estimates fully fit fossil knowledge, past interconnecting bridges between continents, present fasciolid infection in the wild fauna, and lymnaeid distribution. The paleobiogeographical analyses suggest an origin for F. gigantica by transfer from primitive hippopotamuses to grazing bovid ancestors of Reduncinae, Bovinae and Alcelaphinae, by keeping the same vector Radix natalensis in warm lowlands of southeastern Africa in the mid-Miocene, around 13.5 mya. The origin of F. hepatica should have occurred after capture from primitive, less amphibious Hexaprotodon hippopotamuses to mid-sized ovicaprines as the wild bezoar Capra aegagrus and the wild mouflon Ovis gmelini, and from R. natalensis to Galba truncatula in cooler areas and mountainous foothills of Asian Near East in the latest Miocene to Early Pliocene, around 6.0 to 4.0 mya and perhaps shortly afterwards.

Discovery of a Novel Leaf Rust (Puccinia recondita) Resistance Gene in Rye (Secale cereale L.) Using Association Genomics.

Leaf rust constitutes one of the most important foliar diseases in rye (Secale cereale L.). To discover new sources of resistance, we phenotyped 180 lines belonging to a less well-characterized Gülzow germplasm at three field trial locations in Denmark and Northern Germany in 2018 and 2019. We observed lines with high leaf rust resistance efficacy at all locations in both years. A genome-wide association study using 261,406 informative single-nucleotide polymorphisms revealed two genomic regions associated with resistance on chromosome arms 1RS and 7RS, respectively. The most resistance-associated marker on chromosome arm 1RS physically co-localized with molecular markers delimiting Pr3. In the reference genomes Lo7 and Weining, the genomic region associated with resistance on chromosome arm 7RS contained a large number of nucleotide-binding leucine-rich repeat (NLR) genes. Residing in close proximity to the most resistance-associated marker, we identified a cluster of NLRs exhibiting close protein sequence similarity with the wheat leaf rust Lr1 gene situated on chromosome arm 5DL in wheat, which is syntenic to chromosome arm 7RS in rye. Due to the close proximity to the most resistance-associated marker, our findings suggest that the considered leaf rust R gene, provisionally denoted Pr6, could be a Lr1 ortholog in rye.

Phylogenetic and Evolutionary Analysis of the Late Embryogenesis Abundant (LEA) Gene Product in Poaceae

The late Embryogenesis Abundant (LEA) protein family obtains a group of stress-induced hyper-hydrophilic proteins that accumulate in response to cellular dehydration. They are generally unstructured polypeptides without a well-defined three-dimensional structure and have been identified in a wide range of organisms from bacteria to higher plants. Herein, we made a phylogenetic and evolutionary analysis for LEA proteins in Poaceae. The full-length LEA protein sequences were acquired by performing the sequence search of sequenced hva1 against Poaceae species in the non-redundant protein database by a BlastX search tool. The sequences were aligned with the Clustal Omega tool. The MEME suite searched for conserved blocks among each LEA protein sequence. Also, the evolutionary relationship among the LEA protein sequences evaluates using the MEGA tool. The results display close sequence similarity not only into the species but also between species. The results demonstrated that LEA proteins cluster into two large subgroups. The overall average evolutionary difference in LEA protein sequence pairs estimated as 0.4022 amino acid substitutions per site from averaging over all sequence pairs. The LEA protein sequences contain a significant percentage of glycine residues but lack cysteine and tryptophan residues. The results indicate the occurrence of homologs in the subgroup before the divergence of the species. However, the expansion of the gene number in the Poaceae was approved by the duplication events in the preexisting genes rather than by the appearance of the altered LEA gene. Our data will provide novel insights for further studies of the Late Embryogenesis Abundant protein family in Poaceae.

A novel NK-lysin in hybrid crucian carp can exhibit cytotoxic activity in fish cells and confer protection against Aeromonas hydrophila infection in comparison with Carassius cuvieri and Carassius auratus red var

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.

Sporadic isolation of Tetratrichomonas species from the cattle urogenital tract.

Tritrichomonas foetus is a venereal trichomonad parasite which causes reproductive issues in cattle. No other trichomonads are known to be urogenital pathogens in cattle, but there are several reports of Tetratrichomonas and Pentatrichomonas isolates of unclear origin from the cattle urogenital tract (UGT) in the Americas. This study reports the first case of a non-T. foetus cattle urogenital trichomonad isolate in Europe. Molecular analysis of the internal transcribed spacer (ITS) 1-5.8S ribosomal RNA-ITS 2 and 18S ribosomal RNA loci suggest that the isolate is a Tetratrichomonas species from a lineage containing other previously described bull preputial isolates. We identified close sequence similarity between published urogenital and gastrointestinal Tetratrichomonas spp., and this is reviewed alongside further evidence regarding the gastrointestinal origin of non-T. foetus isolates. Routine screening for T. foetus is based on culture and identification by microscopy, and so considering other trichomonad parasites of the bovine UGT is important to avoid misdiagnosis.

Biochemical Characterization of Aminomutases Involved in Salt Tolerance in Methanogenic Archaea

Lysine-2,3-aminomutase (LAM) catalyzes the conversion of L-α-lysine to L-β-lysine, which entails the transfer of an α-amino group to the b-carbon alongside the migration of a β-hydrogen to the α-carbon. This enzyme plays a role in combating salt stress in methanogenic archaea by carrying out the first step in the biosynthesis of Ne-acetyl-β-lysine, a well-studied osmolyte synthesized and accumulated in methanogens. LAM is of special interest as it belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes which perform diverse and complex chemistry that often produces valuable compounds. Radical SAM enzymes utilize a reduced [4Fe-4S] cluster to initiate the homolytic cleavage of SAM, yielding a highly reactive 5’-deoxyadenosyl radical that then abstracts a hydrogen atom from an otherwise unreactive substrate. Although the bacterial LAM involved in lysine degradation in Clostridium subterminale has been well-characterized, the archaeal LAM involved in osmolyte biosynthesis has never been studied in vitro. Here, we report on the recombinant expression, purification, and enzymatic properties of LAM from Methanococcus maripaludis. The gene encoding LAM from M. maripaludis C7 (MmarC7_0106) was cloned and the his-tagged protein was overexpressed in Escherichia coli. After purification by metal-affinity chromatography under strictly anaerobic conditions, the protein was brown in color, had UV-Vis spectrum characteristics of a [4Fe-4S] cluster that was capable of being reduced to an active state, and had 3.9 mol of iron per monomer. A wide ranged temperature profile study revealed that enzymatic activity was highest at 37°C. The enzyme reaction in the presence of L-lysine, SAM, and dithionite at 37°C had a Kcat = 0.42 s-1 and a Km = 17.8 mM. Finally, alternative substrates for the aminomutation reaction were investigated. There exists another gene annotated as LAM in M. maripaludis C7 due to its close sequence similarity with the bacterial LAM; however, this gene (MmarC7_1783) lacks lysine binding residues and has a 164 amino acid N-terminal extension and as such is referred to as a “long-LAM”. The presence of a “long-LAM” such as MmarC7_1783 in a methanogen is not uncommon as there are several annotated in thermophilic methanogenic species that are known to solely use β-glutamate, another beta-amino acid, as their osmolyte. Given that the only known pathway for the biosynthesis of β-glutamate is through the use of a glutamate-2,3-aminomutase (GAM) and that these thermophilic methanogens do not have an annotated GAM, we proposed that these long-LAMs were in fact GAM which we have now confirmed through heterologous expression studies. Thus, in addition to the biochemical characterization of an archaeal LAM (MmarC7_0106) described above, we have determined that long-LAMs are in fact GAMs required in the biosynthesis of an alternative osmolyte used for survival in high salt concentrations by halotolerant methanogenic archaea.

English

Anaplasmosis is a hemo-rickettsial disease of cattle and is most prevalent in tropical and subtropical regions of the world including Pakistan. This disease has been placed as one of the most economically important haemoparasitic diseases. The aim of the current study was to determine the molecular characterization and to assess the prevalence of Anaplasma marginale (A. marginale) infection in cattle and associated risk factors in three districts of Khyber Pakhtunkhwa (KP) province of Pakistan viz., Mardan, Kohat and Swat. The blood samples were collected conveniently from 434 tick-infested animals keeping the aseptic measures. A. marginale was identified from blood samples by microscopy and PCR. Sequencing and phylogenetic analysis of the sequenced isolates of this study showed close sequence similarity with the reported strains of USA, Thailand, Uganda, Uruguay, Zimbabwe, Philippines and China. Moreover, multiple sequence alignment of the 16S ribosomal RNA gene sequences of 5 different clones of the A. marginale depicts substantial variation in the genotypes of A. marginale found in different locations of KP. The prevalence of A. marginale infection was non-significantly associated (P > 0.05) with districts, season, breed, age and sex of cattle. The highest prevalence of A. marginale infection was recorded in district Swat (20.30%) followed by Kohat (16.81%) and Mardan (15.00%) districts of KP. The prevalence of infection was highest in exotic breeds and their crosses, adults and female cattle. 10.70, 16.11, 46.70 and 26.70% were the prevalence of infection recorded for winter, spring, summer and autumn season, respectively. This study concludes that A. marginale infection is dominant in district Swat followed by Kohat and Mardan districts of KP province of Pakistan, respectively. © 2021 Friends Science Publishers

Chimeric ferritin H in hybrid crucian carp exhibits a similar down-regulation in lipopolysaccharide-induced NF-κB inflammatory signal in comparison with Carassius cuvieri and Carassius auratus red var

Ferritin H can participate in the regulation of teleostean immunity. ORF sequences of RCC/WCC/WR-ferritin H were 609 bp, while WR-ferritin H gene possessed chimeric fragments or offspring-specific mutations. In order to elucidate regulation of immune-related signal transduction, three fibroblast-like cell lines derived from caudal fin of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) were characterized and designated as RCCFCs, WCCFCs and WRFCs. A sharp increase of ferritin H mRNA was observed in RCCFCs, WCCFCs and WRFCs following lipopolysaccharide (LPS) challenge. Overexpression of RCC/WCC/WR-ferritin H can decrease MyD88-IRAK4 signal and antagonize NF-κB, TNFα promoter activity in RCCFCs, WCCFCs and WRFCs, respectively. These results indicated that ferritin H in hybrid offspring harbors highly-conserved domains with a close sequence similarity to those of its parents, playing a regulatory role in inflammatory signals.

Sphingomonas horti sp. nov., a novel bacterial species isolated from soil of a tomato garden

A novel bacterial strain, designated MAH-20T, was isolated from a soil sample of a tomato garden. Cells of strain MAH-20T were Gram-stain negative, aerobic, motile, and rod-shaped. The colonies were light brown colored, smooth, spherical, and 0.2-0.7mm in diameter when grown on Luria-Bertani agar for 2days. Strain MAH-20T grows at 15-40°C (optimum growth temperature 30-32°C), at pH 5.0-10.0 (optimum growth pH 7.0) and at 0-2.0% NaCl. The strain showed positive activity for both oxidase and catalase tests. Cells were able to hydrolyze starch, DNA, urea, gelatin, L-arginine, and Tween 20. According to the 16S rRNA gene sequence similarity, the strain MAH-20T was identified as a new member of the genus Sphingomonas and had the close sequence similarity with Sphingomonas changbaiensis V2M44T (98.9%) and Sphingomonas tabacisoli X1-8T (98.1%). The genomic ANI value between strain MAH-20T and S. changbaiensis NBRC 104936T was 84.4%. The novel strain MAH-20T has a draft genome size of 3,350,026bp (25 contigs), annotated with 3210 protein-coding genes, 46 tRNA, and 3 rRNA genes. The genomic DNA G + C content of isolate was 67.3mol%, the predominant quinone was ubiquinone 10 and the major fatty acids were C16:0, C17:1 ω6c and summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c). On the basis of DNA-DNA hybridization results, phenotypic, genotypic, and chemotaxonomic data, the isolated strain MAH-20T represents a novel species, for which the name Sphingomonas horti sp. nov. is proposed, with MAH-20T as the type strain (= KACC 19746T = CGMCC1.13658T).

Identification, molecular characterization, expression analysis and wound-healing ability of multifunctional calreticulin from big-belly seahorse Hippocampus abdominalis

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5′-KHEQSIDCGGGYVKVF-3′ and 5′-LMFGPDICG-3′), triplicate repeats (5′-IKDPEAKKPEDWD-3′, 5′-IPDPDDTKPEDWD-3′, 5′-IPDPDAKKPDDWD-3′), signal peptide and an ER-targeting 5′-KDEL-3′ sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1β (IL-1β). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

CRISPR-mediated gene knockout reveals nicotinic acetylcholine receptor (nAChR) subunit α6 as a target of spinosyns in Helicoverpa armigera.

The spinosyn insecticides (spinosad and spinetoram) have been intensively used to control a wide range of agricultural pests. However, resistance to spinosyns has evolved in several agricultural pests. Disruption of the nicotinic acetylcholine receptor subunit α6 (nAChRα6) has been associated with high levels of resistance to spinosyns in both field and laboratory-selected strains of several insect pests. Among the 12 nAChR subunits of Helicoverpa armigera, Haα6 has the closest sequence similarity (66.02%) to Haα7. Here we used CRISPR-mediated knockouts to evaluate the role of two nAChR subunits (Haα6 and Haα7) of H. armigera in toxicity of spinosyns. Individual knockouts of Haα6 and Haα7 were created utilizing CRISPR/Cas9 system in H. armigera. The Haα6 knockout (Haα6-KO) strain exhibited high levels of resistance to spinosad (531-fold) and spinetoram (1105-fold) compared with the wild-type parent SCD strain, whereas the Haα7 knockout (Haα7-KO) strain showed no significant susceptibility changes to both spinosyns. Genetic analyses demonstrated that resistance to spinosad conferred by knockout of Haα6 was autosomal, incompletely recessive and tightly linked to the disruption mutation of Haα6. Both Haα6-KO and Haα7-KO strains had no significant effects on susceptibility to other four insecticides including emamectin benzoate, beta-cypermethrin, chlorantraniliprole and indoxacarb. Our results provide in vivo functional evidence for Haα6 as a target of spinosyns in H. armigera, and little or no role of Haα7 in mediating toxicity of spinosyns. The results are valuable to the development of resistance monitoring and management methods for spinosyn resistance in H. armigera. © 2020 Society of Chemical Industry.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

Pervasive Differential Splicing in Marek's Disease Virus can Discriminate CVI-988 Vaccine Strain from RB-1B Very Virulent Strain in Chicken Embryonic Fibroblasts.

Marek’s disease is a major scourge challenging poultry health worldwide. It is caused by the highly contagious Marek’s disease virus (MDV), an alphaherpesvirus. Here, we showed that, similar to other members of its Herpesviridae family, MDV also presents a complex landscape of splicing events, most of which are uncharacterised and/or not annotated. Quite strikingly, and although the biological relevance of this fact is unknown, we found that a number of viral splicing isoforms are strain-specific, despite the close sequence similarity of the strains considered: very virulent RB-1B and vaccine CVI-988. We validated our findings by devising an assay that discriminated infections caused by the two strains in chicken embryonic fibroblasts on the basis of the presence of some RNA species. To our knowledge, this study is the first to accomplish such a result, emphasizing how relevant a comprehensive picture of the viral transcriptome is to fully understand viral pathogenesis.

MiR-206 knockout shows it is critical for myogenesis and directly regulates newly identified target mRNAs

ABSTRACT The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.

Toward Development of the Male Pill: A Decade of Potential Non-hormonal Contraceptive Targets.

With the continued steep rise of the global human population, and the paucity of safe and practical contraceptive options available to men, the need for development of effective and reversible non-hormonal methods of male fertility control is widely recognized. Currently there are several contraceptive options available to men, however, none of the non-hormonal alternatives have been clinically approved. To advance progress in the development of a safe and reversible contraceptive for men, further identification of novel reproductive tract-specific druggable protein targets is required. Here we provide an overview of genes/proteins identified in the last decade as specific or highly expressed in the male reproductive tract, with deletion phenotypes leading to complete male infertility in mice. These phenotypes include arrest of spermatogenesis and/or spermiogenesis, abnormal spermiation, abnormal spermatid morphology, abnormal sperm motility, azoospermia, globozoospermia, asthenozoospermia, and/or teratozoospermia, which are all desirable outcomes for a novel male contraceptive. We also consider other associated deletion phenotypes that could impact the desirability of a potential contraceptive. We further discuss novel contraceptive targets underscoring promising leads with the objective of presenting data for potential druggability and whether collateral effects may exist from paralogs with close sequence similarity.

Epidemiological analysis of Arcanobacterium phocae isolated from cases of mink dermatitis of a single farm

The present study was designed to identify nine Arcanobacterium phocae strains isolated from cases of mink dermatitis of a single farm in Finland and characterize the strains for epidemiological relationships. All nine strains and previously described A. phocae used for comparative purposes were identified and further characterized phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), by Fourier Transform Infrared Spectroscopy (FT-IR) and genotypically by detection of phocaelysin encoding gene phl with a previously developed loop-mediated isothermal amplification (LAMP) assay and by sequencing 16S rRNA gene and gene phl, the elongation factor tu encoding gene tuf and the β subunit of bacterial RNA polymerase encoding gene rpoB. Genetic relatedness among isolates was determined using whole-genome single nucleotide polymorphism (wgSNP) analysis. The wgSNP results, partly the MALDI-TOF MS and FT-IR analyses and sequencing of the genes, revealed that the nine A. phocae strains recovered from a single farm showed close sequence similarities among each other and differed from previously investigated A. phocae strains isolated from other farms and animals in Finland and from the A. phocae type strain. This indicated a close epidemiological relationship of the A. phocae strains isolated from a single farm and that the nine A. phocae strains of the present study might have developed from a common ancestor.