Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Phenotypic differentiation between wild and domesticated varieties of <Emphasis Type="Italic">Crescentia cujete</Emphasis>L. and culturally relevant uses of their fruits as bowls in the Yucatan Peninsula,Mexico

Background

Selection criteria are important for analyzing domestication of perennial plant species, which experience a selection pressure throughout several human generations. We analyze the preferred morphological characteristics of Crescentia cujete fruits, which are used as bowls by the Maya of Yucatan, according to the uses they are given and the phenotypic consequences of artificial selection between one wild and three domesticated varieties.

Methods

We performed 40 semi-structured interviews in seven communities. We calculated Sutrop’s salience index (S) of five classes of ceremonial and daily life uses, and of each item from the two most salient classes. We sampled 238 bowls at homes of people interviewed and compared their shape, volume and thickness with 139 fruits collected in homegardens and 179 from the wild. Morphology of varieties was assessed in fruit (n?=?114 trees) and vegetative characters (n?=?136 trees). Differences between varieties were evaluated through linear discriminant analysis (LDA).

Results

Use of bowls as containers for the Day of the Dead offerings was the most salient class (S?=?0.489) with chocolate as its most salient beverage (S?=?0.491), followed by consumption of daily beverages (S?=?0.423), especially maize-based pozol (S?=?0.412). The sacred saka’ and balche' are offered in different sized bowls during agricultural and domestic rituals. Roundness was the most relevant character for these uses, as bowls from households showed a strong selection towards round shapes compared with wild and homegarden fruits. Larger fruits from domesticated varieties were also preferred over small wild fruits, although in the household different sizes of the domesticated varieties are useful. LDA separated wild from domesticated trees (p?<?0.001) according to both fruit and vegetative variables, but domesticated varieties were not different among themselves.

Conclusions

The association between C. cujete bowls and traditional beverages in ritual and daily life situations has driven for centuries the selection of preferred fruit morphology in this tree. Selection of fruit roundness and volume has allowed for the differentiation between the wild variety and the three domesticated ones, counteracting gene flow among them. By choosing the best fruits from domesticated varieties propagated in homegardens, the Maya people model the domestication process of this important tree in their culture.

     

Decrease of insoluble glucan formation in <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by co-cultivation with <Emphasis Type="Italic">Enterococcus faecium</Emphasis> T7 and glucanase addition

Objectives

To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation.

Results

Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium.

Conclusion

E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.

     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

The bacterial community associated with rose-scented geranium (<Emphasis Type="Italic">Pelargonium graveolens</Emphasis>) leaves responds to anthracnose symptoms

Background

The fungus Colletotrichum is a plant pathogen that causes the anthracnose disease, resulting in huge losses in various crops including the rose-scented geranium (Pelargonium graveolens). Although the bacterial community associated with plants has an important role in the establishment of plant diseases, little is known about what happens in P. graveolens.

Aims

To increase the knowledge about the bacterial community associated with P. graveolens and its relationship with anthracnose disease symptoms.

Methods

Quantitative PCR and high-throughput sequencing were combined to determine the presence of the fungus Colletotrichum and to reveal the bacterial communities associated with different plant parts – root, stem and leaf – and in the rhizosphere and bulk soil, and also to determine the respective bacterial communities associated with P. graveolens leaves symptomatic and asymptomatic for anthracnose disease.

Results

The fungus Colletotrichum was detected in all plant parts and in the surrounding soil. Bacterial communities varied spatially in plants, and the disease symptoms also influenced the composition of the bacterial community. Abundances of operational taxonomic units (OTUs) assigned to the phylum Actinobacteria and to the genus Streptococcus were greatly increased in asymptomatic leaves.

Conclusions

The bacterial community associated to geranium leaves responds to anthracnose symptoms.

     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

Adjunctive corticosteroid therapy for inpatients with <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> pneumonia

Background

There is conflicting evidence regarding the benefit of adjunctive corticosteroid therapy in patients with Mycoplasma pneumoniae pneumonia. We hypothesised that corticosteroid therapy could reduce mortality and length of stay (LOS) in such patients.

Methods

Adult patients with M. pneumoniae pneumonia from January 2010 to December 2013 were identified from the Japanese Diagnosis Procedure Combination inpatient database. The effects of low-dose and high-dose corticosteroid therapies on mortality, LOS, drug costs and hyperglycaemia requiring insulin treatment were evaluated using propensity score analyses.

Results

Eligible patients (n?=?2228) from 630 hospitals were divided into no-corticosteroid (n?=?1829), low-dose corticosteroid (n?=?267) and high-dose corticosteroid (n?=?132) groups. The propensity score-matched pairs were generated from no-corticoid and low-dose corticoid groups (251 pairs), or no-corticoid and high-dose corticosteroid groups (120 pairs). Adjunctive corticosteroid therapy did not decrease 30-day mortality. In addition, both low-dose and high-dose corticosteroid therapies were associated with increases in LOS. Furthermore, hyperglycaemia requiring insulin treatment and drug cost increased with corticosteroid use.

Conclusions

Adjunctive treatment with low-dose or high-dose corticosteroids may not be beneficial in M. pneumoniae pneumonia.

     

Metabolomics reveals immunomodulation as a possible mechanism for the antibiotic effect of <Emphasis Type="Italic">Persicaria capitata</Emphasis> (Buch.-Ham. ex D. Don) H.Gross

Introduction

In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.

Objective

The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.

Methods

Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).

Results

The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.

Conclusion

Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.

     

Enhanced production of fructosyltransferase in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> by genome shuffling

Objective

To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling.

Results

A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180 U g^?1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 U g^?1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201.

Conclusions

Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Effects of <Emphasis Type="Italic">ADIPOQ</Emphasis> polymorphisms on PCOS risk: a meta-analysis

Background

Whether adiponectin (ADIPOQ) polymorphisms are associated with the risk of polycystic ovary syndrome (PCOS) remain controversial. Therefore, we performed this study to better explore correlations between ADIPOQ polymorphisms and PCOS risk.

Methods

Literature retrieve was conducted in PubMed, Medline and Embase. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated.

Results

Eighteen studies were enrolled for analyses. Pooled overall analyses showed that rs1501299 polymorphism was significantly associated with PCOS risk (recessive model: p?=?0.02, OR?=?0.77, 95%CI 0.62–0.95; allele model: p?=?0.001, OR?=?1.15, 95%CI 1.06–1.26). Further subgroup analyses according to ethnicity of participants revealed that rs1501299 and rs2241766 polymorphisms were both significantly correlated with PCOS risk in Caucasians. In addition, rs1501299 polymorphism was also significantly correlated with PCOS risk in East Asians.

Conclusions

Our findings indicated that rs1501299 and rs2241766 polymorphisms might serve as genetic biomarkers of PCOS in certain ethnicities.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Ethnomedicines and anti-parasitic activities of Pakistani medicinal plants against <Emphasis Type="Italic">Plasmodia</Emphasis> and <Emphasis Type="Italic">Leishmania</Emphasis> parasites

Background

Leishmaniasis and malaria are the two most common parasitic diseases and responsible for large number of deaths per year particularly in developing countries like Pakistan. Majority of Pakistan population rely on medicinal plants due to their low socio-economic status. The present review was designed to gather utmost fragmented published data on traditionally used medicinal plants against leishmaniasis and malaria in Pakistan and their scientific validation.

Methods

Pub Med, Google Scholar, Web of Science, ISI Web of knowledge and Flora of Pakistan were searched for the collection of data on ethnomedicinal plants. Total 89 articles were reviewed for present study which was mostly published in English. We selected only those articles in which complete information was given regarding traditional uses of medicinal plants in Pakistan.

Results

Total of 56 plants (malaria 33, leishmaniasis 23) was found to be used traditionally against reported parasites. Leaves were the most focused plant part both in traditional use and in in vitro screening against both parasites. Most extensively used plant families against Leishmaniasis and Malaria were Lamiaceae and Asteraceae respectively. Out of 56 documented plants only 15 plants (Plasmodia 4, Leishmania 11) were assessed in vitro against these parasites. Mostly crude and ethanolic plant extracts were checked against Leishmania and Plasmodia respectively and showed good inhibition zone. Four pure compounds like artemisinin, physalins and sitosterol extracted from different plants proved their efficacy against these parasites.

Conclusions

Present review provides the efficacy and reliability of ethnomedicinal practices and also invites the attention of chemists, pharmacologist and pharmacist to scientifically validate unexplored plants that could lead toward the development of novel anti-malarial and anti-leishmanial drugs.