Examination of unidentifiable spined loach individuals found in the overlap zone of two tetraploid species within a single river in Japan

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them.     

In silico identification of novel lncRNAs with a potential role in diagnosis of gastric cancer

Abstract

Gastric cancer (GC) is the second leading cause of cancer-related deaths in the world. Due to the shortage of adequate symptoms in the early stages, it is diagnosed when the tumor has spread to distant organs. Early recognition of GC enhances the chance of successful treatment. Molecular mechanisms of GC are still poorly understood. LncRNAs are emerging as new players in cancer in both oncogene and tumor suppressor roles. High-throughput technologies such as RNA-Seq, have revealed thousands of lncRNAs which are dysregulated in GC. In this study, we retrieved lncRNAs obtained by High-throughput technologies from OncoLnc database. Consequently, retrieved lncRNAs were compared in literature-based databases including PubMed. As a result, two lists, including experimentally validated lncRNAs and predicted lncRNAs were provided. We found 43 predicted lncRNAs that had not been experimentally validated in GC, so far. Further Bioinformatics analyses were performed to obtain the expression profile of predicted lncRNAs in tumor and normal tissues. Also, the roles and targets of predicted lncRNAs in GC were identified by related databases. Finally, using the GEPIA database was reviewed the significant relationship of predicted lncRNAs with the survival of GC patients. By recognizing the lncRNAs involved in initiation and progression of GC, they may be considered as potential biomarkers in the GC early diagnosis or targeted treatment and lead to novel therapeutic strategies.

Communicated by Ramaswamy H. Sarma     

Cryopreservation of shoot tips,evaluations of vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.     

Real-time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control

Dissolved carbon dioxide (dCO₂) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO₂ in bioprocesses. A silicone sampling probe that allows the diffusion of CO₂ through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO₂ that had diffused into the probe from the culture broth (sensor was calibrated using air as zero-point calibration). The gas inside the probe was then allowed to recirculate through gas-impermeable tubing to a CO₂ monitor. We have shown that by measuring the initial diffusion rate of CO₂ into the sampling probe we were able to determine the partial pressure of the dCO₂ in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO₂ in real-time. Using the proposed sensor, we successfully implemented a dCO₂-based control scheme, which resulted in significant improvement in process performance.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya,India

Importance to know and understand diversity of Himalayan plants is increasingly recognized considering the fact that various natural and anthropogenic pressures might bring about serious influences to morphological and genetic diversity of the vegetation in the region. In this context, Valeriana jatamansi was investigated in detail, taking into account its importance in various Ayurvedic and modern medicines. Randomly selected mature plants from twenty five different populations (located between 1215 m to 2775 m asl) of V. jatamansi were analysed for their morphological attributes. Further, ISSR markers were used to detect genetic variation among 151 plants of selected 25 populations. Use of 20 primers yielded 125 reproducible polymorphic loci which were used to estimate different parameters of genetic diversity. These parameters were in turn applied to develop relationships with habitat types and altitude range. Significant variation (p < 0.05) in above ground dry weight (AGDW) and below ground dry weight (BGDW) across the populations was observed. Nei's genetic diversity index (He) ranged from 0.25 to 0.37 across the populations, with a mean of 0.31. Genetic diversity exhibited a decreasing trend with increasing altitude, and maximum diversity (He = 0.325) was observed in the range of 1201–1500 m asl. Among the different habitat conditions, highest genetic diversity (He = 0.334; Pp = 84.38) was observed in grassland habitats while minimum in mixed forest habitats (He = 0.285; Pp = 72.433). The genetic diversity (He) had significant negative relationships with AGDW, BGDW and rhizome diameter (Pearson r = −0.359, −0.424 and −0.317, respectively; p < 0.05). The genetic characterization of V. jatamansi from the western Himalaya by this study suggests influences of habitat types and the altitudinal range upon genetic diversity, and based on these proposals for conservation strategies in favour of the species are made.     

Characterization of the first adult de novo case of 46,X,der(Y)t(X;Y)(p22.3;q11.2)

Herein, we describe a case of an infertile man detected in postnatal diagnosis with FISH characterization and array-CGH used for genome-wide screening which allowed the identification of a complex rearrangement involving sex chromosomes, apparently without severe phenotypic consequences. The deletion detected in our patient has been compared with previously reported cases leading us to propose a hypothetical diagnostic algorithm that would be useful in similar clinical situations, with imperative multi disciplinary approach integrated with genetic counseling. Our patient, uniquely of reproductive age, is one of six reported cases of duplication of Xp22.3 (~ 8.4 Mb) segment and contemporary deletion of Yq (~ 42.9 Mb) with final karyotype as follows:

46,X,der(Y),t(X;Y)(Ypter → Yq11.221::Xp22.33 → Xpter).ish der(Y) (Yptel+,Ycen+,RP11-529I21+,RP11-506M9-Yqtel −,Xptel +). arrXp22.33p22.31(702–8,395,963, 8,408,289x1), Yq11.221q12 (14,569,317x1, 14,587,321–57,440,839x0)     

Nanocapsules: A Novel Lipid Formulation Platform for Platinum-based Anti-cancer Drugs

Platinum-based anti-cancer agents have been used for many years to treat many different types of cancer. However, the efficacy of these drugs is limited by serious side effects. One of the strategies to reduce the side effects is encapsulation of the drug in a lipid formulation. Recently, we discovered a novel method for the efficient encapsulation of cisplatin in a lipid formulation. The method is unique in that it does not generate conventional liposomes but nanocapsules: small aggregates of solid cisplatin covered by a lipid bilayer. Also carboplatin, a cisplatin-derived anti-cancer drug with different chemical properties, can be efficiently encapsulated by a similar method. The encapsulation in nanocapsules dramatically improves the in vitro cytotoxicity of the platinum drugs. Our results hold the promise that the nanocapsule technology could prove successful in the efficient encapsulation of many other (platinum-based) drugs, and thereby improve their therapeutic index and profile in vivo.