ResearchPad - extraction-techniques Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Insight into the protein solubility driving forces with neural attention]]> The solubility of proteins is a crucial biophysical aspect when it comes to understanding many human diseases and to improve the industrial processes for protein production. Due to its relevance, computational methods have been devised in order to study and possibly optimize the solubility of proteins. In this work we apply a deep-learning technique, called neural attention to predict protein solubility while “opening” the model itself to interpretability, even though Machine Learning models are usually considered black boxes. Thank to the attention mechanism, we show that i) our model implicitly learns complex patterns related to emergent, protein folding-related, aspects such as to recognize β-amyloidosis regions and that ii) the N-and C-termini are the regions with the highes signal fro solubility prediction. When it comes to enhancing the solubility of proteins, we, for the first time, propose to investigate the synergistic effects of tandem mutations instead of “single” mutations, suggesting that this could minimize the number of required proposed mutations.

<![CDATA[Genetic algorithm-based personalized models of human cardiac action potential]]> We present a novel modification of genetic algorithm (GA) which determines personalized parameters of cardiomyocyte electrophysiology model based on set of experimental human action potential (AP) recorded at different heart rates. In order to find the steady state solution, the optimized algorithm performs simultaneous search in the parametric and slow variables spaces. We demonstrate that several GA modifications are required for effective convergence. Firstly, we used Cauchy mutation along a random direction in the parametric space. Secondly, relatively large number of elite organisms (6–10% of the population passed on to new generation) was required for effective convergence. Test runs with synthetic AP as input data indicate that algorithm error is low for high amplitude ionic currents (1.6±1.6% for IKr, 3.2±3.5% for IK1, 3.9±3.5% for INa, 8.2±6.3% for ICaL). Experimental signal-to-noise ratio above 28 dB was required for high quality GA performance. GA was validated against optical mapping recordings of human ventricular AP and mRNA expression profile of donor hearts. In particular, GA output parameters were rescaled proportionally to mRNA levels ratio between patients. We have demonstrated that mRNA-based models predict the AP waveform dependence on heart rate with high precision. The latter also provides a novel technique of model personalization that makes it possible to map gene expression profile to cardiac function.

<![CDATA[NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies]]>

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer’s disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders.

<![CDATA[Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella]]>

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay’s specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.

<![CDATA[Advanced approach to analyzing calcareous protists for present and past pelagic ecology: Comprehensive analysis of 3D-morphology, stable isotopes, and genes of planktic foraminifers]]>

Marine protists play an important role in oceanic ecosystems and biogeochemical cycles. However, the difficulties in culturing pelagic protists indicate that their ecology and behavior remain poorly understood; phylogeographic studies based on single-cell genetic analyses have often shown that they are highly divergent at the biological species level, with variable geographic distributions. This indicates that their ecology could be complex. On the other hand, the biomineral (calcareous) shells of planktic foraminifers are widely used in geochemical analyses to estimate marine paleoenvironmental characteristics (i.e., temperature), because the shell chemical composition reflects ambient seawater conditions. Among the pelagic protists, planktic foraminifers are ideal study candidates to develop a combined approach of genetic, morphological, and geochemical methods, thus reflecting environmental and ecological characteristics. The present study precisely tested whether the DNA extraction process physically and chemically affects the shells of the planktic foraminifer Globigerinoides ruber. We used a nondestructive method for analyzing physical changes (micro-focus X-ray computed tomography (MXCT) scanning) to compare specimens at the pre- and post-DNA extraction stages. Our results demonstrate that DNA extraction has no significant effect on shell density and thickness. We measured stable carbon and oxygen isotopes on the shell of each individual in a negative control or one of two DNA-extracted groups and detected no significant differences in isotopic values among the three groups. Moreover, we evaluated isotopic variations at the biological species level with regard to their ecological characteristics such as depth habitat, life stages, and symbionts. Thus, our examination of the physiochemical effects on biomineral shells through DNA extraction shows that morphological and isotopic analyses of foraminifers can be combined with genetic analysis. These analytical methods are applicable to other shell-forming protists and microorganisms. In this study, we developed a powerful analytical tool for use in ecological and environmental studies of modern and past oceans.

<![CDATA[A survey on Mycobacterium ulcerans in Mosquitoes and March flies captured from endemic areas of Northern Queensland, Australia]]>

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). This nontuberculous mycobacterial infection has been reported in 34 countries worldwide. In Australia, the majority of cases of BU have been recorded in coastal Victoria and the Mossman-Daintree areas of north Queensland. Mosquitoes have been postulated as a vector of M. ulcerans in Victoria, however the specific mode of transmission of this disease is still far from being well understood. In the current study, we trapped and analysed 16,900 (allocated to 845 pools) mosquitoes and 296 March flies from the endemic areas of north Queensland to examine for the presence of M. ulcerans DNA by polymerase chain reaction. Seven of 845 pools of mosquitoes were positive on screening using the IS2404 PCR target (maximum likelihood estimate 0.4/1,000). M. ulcerans DNA was detected from one pool of mosquitoes from which all three PCR targets: IS2404, IS2606 and the ketoreductase B domain of mycolactone polyketide synthase gene were detected. None of the March fly samples were positive for the presence of M. ulcerans DNA.

<![CDATA[Prevalence of infection by the microsporidian Nosema spp. in native bumblebees (Bombus spp.) in northern Thailand]]>

Bumblebees (tribe Bombini, genus Bombus Latreille) play a pivotal role as pollinators in mountain regions for both native plants and for agricultural systems. In our survey of northern Thailand, four species of bumblebees (Bombus (Megabombus) montivagus Smith, B. (Alpigenobombus) breviceps Smith, B. (Orientalibombus) haemorrhoidalis Smith and B. (Melanobombus) eximius Smith), were present in 11 localities in 4 provinces (Chiang Mai, Mae Hong Son, Chiang Rai and Nan). We collected and screened 280 foraging worker bumblebees for microsporidia (Nosema spp.) and trypanosomes (Crithidia spp.). Our study is the first to demonstrate the parasite infection in bumblebees in northern Thailand. We found N. ceranae in B. montivagus (5.35%), B. haemorrhoidalis (4.76%), and B. breviceps (14.28%) and N. bombi in B. montivagus (14.28%), B. haemorrhoidalis (11.64%), and B. breviceps (28.257%).

<![CDATA[Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein]]>

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.

<![CDATA[A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib]]>

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

<![CDATA[Comparison of the new fully automated extraction platform eMAG to the MagNA PURE 96 and the well-established easyMAG for detection of common human respiratory viruses]]>

Respiratory viral infections constitute the majority of samples tested in the clinical virology laboratory during the winter season, and are mainly diagnosed using molecular assays, namely real-time PCR (qPCR). Therefore, a high-quality extraction process is critical for successful, reliable and sensitive qPCR results. Here we aimed to evaluate the performance of the newly launched eMAG compared to the fully automated MagNA PURE 96 (Roche, Germany) and to the semi-automated easyMAG (bioMerieux, France) extraction platforms. For this analysis, we assessed and compared the analytic and clinical performance of the three platforms, using 262 archived respiratory samples positive or negative to common viruses regularly examined in our laboratory (influenza A, B, H1N1pdm, Respiratory Syncytial Virus (RSV), human Metapneumovirus (hMPV), parainfluenza-3, adenovirus and negative samples). In addition, quantitated virus controls were used to determine the limit of detection of each extraction method.

In all categories tested, eMAG results were comparable to those of the easyMAG and MagNa PURE 96, highly sensitive for all viruses and over 98% clinical specificity and sensitivity for all viruses tested. Together with its high level of automation, the bioMerieux eMAG is a high-quality extraction platform enabling effective molecular analysis and is mostly suitable for medium-sized laboratories.

<![CDATA[Multiplexing polysome profiling experiments to study translation in Escherichia coli]]>

Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.

<![CDATA[O-GlcNAcylation of PERIOD regulates its interaction with CLOCK and timing of circadian transcriptional repression]]>

Circadian clocks coordinate time-of-day-specific metabolic and physiological processes to maximize organismal performance and fitness. In addition to light and temperature, which are regarded as strong zeitgebers for circadian clock entrainment, metabolic input has now emerged as an important signal for clock entrainment and modulation. Circadian clock proteins have been identified to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs is now recognized as an important mechanism by which metabolic input regulates circadian physiology. To better understand the role of O-GlcNAcylation in modulating clock protein function within the molecular oscillator, we used mass spectrometry proteomics to identify O-GlcNAcylation sites of PERIOD (PER), a repressor of the circadian transcriptome and a critical biochemical timer of the Drosophila clock. In vivo functional characterization of PER O-GlcNAcylation sites indicates that O-GlcNAcylation at PER(S942) reduces interactions between PER and CLOCK (CLK), the key transcriptional activator of clock-controlled genes. Since we observe a correlation between clock-controlled daytime feeding activity and higher level of PER O-GlcNAcylation, we propose that PER(S942) O-GlcNAcylation during the day functions to prevent premature initiation of circadian repression phase. This is consistent with the period-shortening behavioral phenotype of per(S942A) flies. Taken together, our results support that clock-controlled feeding activity provides metabolic signals to reinforce light entrainment to regulate circadian physiology at the post-translational level. The interplay between O-GlcNAcylation and other PTMs to regulate circadian physiology is expected to be complex and extensive, and reach far beyond the molecular oscillator.

<![CDATA[Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation]]>

Hbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

<![CDATA[How to integrate wet lab and bioinformatics procedures for wine DNA admixture analysis and compositional profiling: Case studies and perspectives]]>

The varietal authentication of wines is fundamental for assessing wine quality, and it is part of its compositional profiling. The availability of historical, cultural and chemical composition information is extremely important for quality evaluation. DNA-based techniques are a powerful tool for proving the varietal composition of a wine. SSR-amplification of genomic residual Vitis vinifera DNA, namely Wine DNA Fingerprinting (WDF) is able to produce strong, analytical evidence concerning the monovarietal nature of a wine, and for blended wines by generating the probability of the presence/absence of a certain variety, all in association with a dedicated bioinformatics elaboration of genotypes associated with possible varietal candidates. Together with WDF we could exploit Bioinformatics techniques, due to the number of grape genomes grown. In this paper, the use of WDF and the development of a bioinformatics tool for allelic data validation, retrieved from the amplification of 7 to 10 SSRs markers in the Vitis vinifera genome, are reported. The wines were chosen based on increasing complexity; from monovarietal, experimental ones, to commercial monovarietals, to blended commercial wines. The results demonstrate that WDF, after calculation of different distance matrices and Neighbor-Joining input data, followed by Principal Component Analysis (PCA) can effectively describe the varietal nature of wines. In the unknown blended wines the WDF profiles were compared to possible varietal candidates (Merlot, Pinot Noir, Cabernet Sauvignon and Zinfandel), and the output graphs show the most probable varieties used in the blend as closeness to the tested wine. This pioneering work should be meant as to favor in perspective the multidisciplinary building-up of on-line databanks and bioinformatics toolkits on wine. The paper concludes with a discussion on an integrated decision support system based on bioinformatics, chemistry and cultural data to assess wine quality.

<![CDATA[Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription]]>

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

<![CDATA[Virus load and clinical features during the acute phase of Chikungunya infection in children]]>


Chikungunya virus (CHIKV) infection is a long known mosquito-borne disease that is associated with severe morbidity, characterized by fever, headache, rashes, joint pain, and myalgia. It is believed that virus load has relation with severity of clinical features.


We performed this study to assess the relationship between virus load and clinical features in children during the acute phase of CHIKV infection, in order to draw insights for better-informed treatment.

Study design

Between June 1, 2009, and May 31, 2010, 338 patients with fever and susceptive to CHIKV during first 4 days of illness were prospectively enrolled from Karnataka Institute of Medical Sciences, Hubli in our hospital based cross sectional observational study. Sybr green quantitative reverse transcription polymerase chain reaction was performed to estimate the virus load.


Quantitative RT-PCR was positive for CHIKV in 54 patients. The median copy number of CHIKV was 1.3x 108 copies/ml (1.7x105-9.9x109 copies/ml). Among the observed clinical features, a statistically significant difference in log mean virus load was found between patients with and without myalgia (log mean 7.50 vs 8.34, P = 0.01).


Patients with myalgia had lower virus load and those without myalgia had a higher virus load.

<![CDATA[Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms]]>

Tomato yellow leaf curl virus (TYLCV) and its related begomoviruses cause fast-spreading diseases in tomato worldwide. How this virus induces diseases remains largely unclear. Here we report a noncoding RNA-mediated model to elucidate the molecular mechanisms of TYLCV-tomato interaction and disease development. The circular ssDNA genome of TYLCV contains a noncoding intergenic region (IR), which is known to mediate viral DNA replication and transcription in host cells, but has not been reported to contribute directly to viral disease development. We demonstrate that the IR is transcribed in dual orientations during plant infection and confers abnormal phenotypes in tomato independently of protein-coding regions of the viral genome. We show that the IR sequence has a 25-nt segment that is almost perfectly complementary to a long noncoding RNA (lncRNA, designated as SlLNR1) in TYLCV-susceptible tomato cultivars but not in resistant cultivars which contains a 14-nt deletion in the 25-nt region. Consequently, we show that viral small-interfering RNAs (vsRNAs) derived from the 25-nt IR sequence induces silencing of SlLNR1 in susceptible tomato plants but not resistant plants, and this SlLNR1 downregulation is associated with stunted and curled leaf phenotypes reminiscent of TYLCV symptoms. These results suggest that the lncRNA interacts with the IR-derived vsRNAs to control disease development during TYLCV infection. Consistent with its possible function in virus disease development, over-expression of SlLNR1 in tomato reduces the accumulation of TYLCV. Furthermore, gene silencing of the SlLNR1 in the tomato plants induced TYLCV-like leaf phenotypes without viral infection. Our results uncover a previously unknown interaction between vsRNAs and host lncRNA, and provide a plausible model for TYLCV-induced diseases and host antiviral immunity, which would help to develop effective strategies for the control of this important viral pathogen.

<![CDATA[Thermal acclimation of photosynthetic activity and RuBisCO content in two hybrid poplar clones]]>

The mechanistic bases of thermal acclimation of net photosynthetic rate (An) are still difficult to discern, and the data sets available are scarce, particularly for hybrid poplar. In the present study, we examined the contribution of a number of biochemical and biophysical traits on thermal acclimation of An for two hybrid poplar clones. We grew cuttings of Populus maximowiczii × Populus nigra (M×N) and Populus maximowiczii × Populus balsamifera (M×B) clones under two day/night temperature of 23°C/18°C and 33°C /27°C and under low and high soil nitrogen level. After ten weeks, we measured leaf RuBisCO (RAR) and RuBisCO activase (RARCA) amounts and the temperature response of An, dark respiration (Rd), stomatal conductance, (gs), apparent maximum carboxylation rate of CO2 (Vcmax) and apparent photosynthetic electron transport rate (J). Results showed that a 10°C increase in growth temperature resulted in a shift in thermal optimum (Topt) of An of 6.2±1.6°C and 8.0±1.2°C for clone M×B and M×N respectively, and an increased An and gs at the growth temperature for clone M×B but not M×N. RuBisCO amount was increased by N level but was insensitive to growth temperature while RARCA amount and the ratio of its short to long isoform was stimulated by the warm condition for clone M×N and at low N for clone M×B. The activation energy of apparent Vcmax and apparent J decreased under the warm condition for clone M×B and remained unchanged for clone M×N. Our study demonstrated the involvement of both RARCA, the activation energy of apparent Vcmax and stomatal conductance in thermal acclimation of An.

<![CDATA[Viable Neisseria meningitidis is commonly present in saliva in healthy young adults: Non-invasive sampling and enhanced sensitivity of detection in a follow-up carriage study in Portuguese students]]>

Introduction and aims

Improved sensitivity and efficiency of detection and quantification of carriage of Neisseria meningitidis (Nm) in young people is important for evaluation of the impact of vaccines upon transmission and associated population-wide effects. Saliva collection is quick, non-invasive and facilitates frequent sampling, but has been reported to yield low sensitivity by culture. We re-evaluated this approach in a follow-up cross sectional study using direct and culture-amplified PCR.


In April 2016 we collected paired oropharyngeal swabs (OPS) and saliva samples from 1005 healthy students in Portugal into STGG broth and stored them at -80°C until DNA extraction and batched qPCR analysis. Samples were also cultured on GC agar plates for 72h and PCR done on DNA extracts from overall growth. Nm isolates were also sought from a selection of 50 samples. qPCR amplification targets were superoxide dismutase sodC and capsular locus/genogroup-specific genes (B, C, W, X and Y) and, for cultured isolates only, porA. Cycle threshold values of ≤36 were considered positive.


556 tests (460 samples, 363 subjects, 36.1%) were positive for Nm (sodC) and 65 (45, 36, 3.6%) for MenB. More salivas were positive by direct sodC qPCR (211, 21.0%) than OPS (126, 12.5%) but fewer were positive by culture-amplified qPCR (94 vs. 125). For both sample types, many that were negative on direct qPCR came positive on culture-amplification and Nm was consistently isolated from salivas in which culture amplified the PCR signal. Using both methods on both samples yielded 36.1% Nm and 5.5% encapsulated Nm carriage rates while direct qPCR on OPS alone detected 12.5% and 2.2%.


Detectable MenB carriage rates (2.9%) were lower than 4 years earlier (6.8%) in this population (p = 0.0003). Viable meningococci were often present in saliva. Although evidence of encapsulated Nm was less frequent in saliva than OPS, collection is more acceptable to subjects allowing more frequent sampling. Use of culture-amplification increases detection sensitivity in both sample types, especially when combined with direct PCR. Combining these samples and/or methodologies could greatly enhance the power of carriage studies to detect the impact of vaccines upon carriage and transmission.

<![CDATA[Quality and quantity of dromedary camel DNA sampled from whole-blood, saliva, and tail-hair]]>

Camels are livestock with unique adaptations to hot-arid regions. To effectively study camel traits, a biobank of camel DNA specimens with associated biological information is needed. We examined whole-blood, saliva (buccal swabs), and tail-hair follicle samples to determine which is the best source for establishing a DNA biobank. We inspected five amounts of each of whole-blood, buccal swabs, and tail-hair follicles in nine camels, both qualitatively via gel electrophoresis and quantitatively using a NanoDrop spectrophotometer. We also tested the effects of long term-storage on the quality and quantity of DNA, and measured the rate of degradation, by analyzing three buccal swab samples and 30 tail-hair follicles over a period of nine months. Good quality DNA, in the form of visible large size DNA bands, was extracted from all three sources, for all five amounts. The five volumes of whole-blood samples (20–100μl) provided ~0.4–3.6 μg, the five quantities of buccal swabs (1–5) produced ~0.1–12 μg, while the five amounts of tail-hair follicles (10–50) resulted in ~0.7–25 μg. No differences in the rate of degradation of buccal swab and tail-hair follicle DNA were detected, but there was clearly greater deterioration in the quality of DNA extracted from buccal swabs when compared to tail-hair follicles. We recommend using tail-hair samples for camel DNA biobanking, because it resulted in both an adequate quality and quantity of DNA, along with its ease of collection, transportation, and storage. Compared to its success in studies of other domesticated animals, we anticipate that using ~50 tail-hair follicles will provide sufficient DNA for sequencing or SNP genotyping.