Asian Crocodile Poaching:A Review of Molecular Techniques Developed for Forensic Identification

Crocodiles,gharials and alligators(order Crocodilia),are aquatic reptiles that live in the tropics of Asia,America,Africa,and Australia.Asian countries such as India,Indonesia,Malaysia,and tropics of Australia are the stronghold of the family Crocodylidae.Among all 23 crocodile species,nine species occur in Asia and its surroundings,including the only member of Gavialidae and Alligatoridae family.They are“mugger”or“Crocodylus palustris,”“saltwater crocodile”or“Crocodylus porosus,”“Philippine crocodile”or“Crocodylus mindorensis,”“New Guinea crocodile”or“Crocodylus novaeguineae,”“Siamese crocodile”or“Crocodylus siamensis,”“gharials”or“Gavialis gangeticus,”“false gharial”or“Tomistoma schlegelii,”and“Chinese alligator”or“Alligator sinensis.”All of these species have been encompassed in“Appendix I”and“Appendix II”of the“Convention on International Trade in Endangered Species of Wild Fauna and Flora,”which prevents any kind of trade involving crocodilian species.However,it has been observed that these crocodiles are illegally poached and trafficked for their lucrative skin,meats,eggs,snouts,and bones in medicinal and cosmetic industries.Although many molecular biologists have come forward for the conservation of these species,lack of knowledge about the available,fast,and dependable techniques makes it difficult for forensic identification of seized or confiscated.It has been a major problem for the implementation of the“Wildlife Protection Law”on illegal trade.This article focuses on molecular techniques developed till date for the rapid and reliable species identification and conservation study of them.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

Clinical significance of molecular diagnosis for gastric cancer lymph node micrometastasis

Advances in molecular diagnostic tools have allowed the identification of lymph node micrometastasis(LNM),including isolated tumor cells,in cancer patients. While immunohistochemistry and reverse transcription-polymerase chain reaction have been used to identify LNM in patients with gastric cancer,the clinical significance of this finding remains unclear. Recently,minimally invasive treatments,such as endoscopic submucosal dissection and laparoscopic surgery,are widely performed to help improve postsurgical quality of life(QOL). However,it is important to maintain the balance between QOL and curability when making treatments decision for patients with gastric cancer. If minimally invasive surgery based on accurate intraoperative LNM diagnosis was established,it could be performed safely. Therefore,we reviewed the clinical significance of LNM detected by molecular techniques as an important target for treatment decision making with gastric cancer patients.

Molecular diagnosis and therapy for occult peritoneal metastasis in gastric cancer patients

To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process,denoting metastatic disease.The accuracy of cytological diagnosis leaves room for improvement;therefore,highly sensitive molecular diagnostics,such as an enzyme immunoassay,reverse transcription polymerase chain reaction,and virusguided imaging,have been developed to detect minute cancer cells in the peritoneal cavity.Molecular targeting therapy has also been spun off from basic research in the past decade.Although conventional cytologyis still the mainstay,novel approaches could serve as practical complementary diagnostics to cytology in near future.

Biocompatibility evaluation in vitro. Part III: Cytotoxicity expression of human and animal osteoblasts on the biomaterials

Exclusion and inflammation in the clinic are observe d for various reas ons including material che- mical composition, physical properties as well a s macr o- and micro-structure of the implants, surface condition of the implants, and also patient dependent factors. Cytotoxicity expression of cells is a central issue i n current biocompatibility to screen the potential implant materials and drugs. This study was aimed at investigation reaction between the potential implant mat erials and surround tissue. Cytotoxicity of human and rat osteoblast in the mate rial extracts was determinated by testing standards such as GHS assay, MTT assay , alkaline phosphatase activity assay, LDH assay, and Lowry assay. Research resu lts demonstrated that compared with the control condition polystyrene culture pl a te both human and rat osteoblast cells have normal phenotypic expression in hydr oxyapatite extract, and this expression was statistically restricted in hydroxya patite-spinel extract. How- ever, this restrict, e.g. cytotoxicity could be partially eliminated by immersion treatment of the materials in culture medium.

Analysis of Microbial Molecular Ecology Techniques in Constructed Rapid Infiltration System

The microbial molecular ecology techniques, which were developed on the basis of molecular, were applied in studying the bacteria in Constructed Rapid Infiltration （CRI） system. These techniques are very efficient in better describing the bacterial diversity, microbial community distribution, and relations between microbial group structure and nitrogen contamination through the analysis of microbial nucleic acid sequence fragment in CRI. The results further revealed the removal mechanism of contamination, which is essential for the improvement of wastewater treatment in CRI.

Current molecular biologic techniques for characterizing environmental microbial community

Microbes are vital to the earth because of their enormous numbers and instinct function maintaining the natural balance. Since the microbiology was applied in environmental science and engineering more than a century ago, researchers desire for more and more information concerning the microbial spatio-temporal variations in almost every fields from contaminated soil to wastewater treatment plant （WWTP）. For the past 30 years, molecular biologic techniques explored for environ- mental microbial community （EMC） have spanned a broad range of approaches to facilitate the researches with the assistance of computer science： faster, more accurate and more sensitive. In this feature article, we outlined several current and emerging molecular biologic techniques applied in detection of EMC, and presented and assessed in detail the application of three promising tools.

Detection of Helicobacter pylori resistance to clarithromycin and fluoroquinolones in Brazil:A national survey

AIM To evaluate bacterial resistance to clarithromycin and fluoroquinolones in Brazil using molecular methods.METHODS The primary antibiotic resistance rates of Helicobacter pylori(H. pylori) were determined from November 2012 to March 2015 in the Southern,South-Eastern,Northern,North-Eastern,and Central-Western regions of Brazil. Four hundred ninety H. pylori patients [66% female,mean age 43 years(range: 18-79)] who had never been previously treated for this infection were enrolled. All patients underwent gastroscopy with antrum and corpus biopsies and molecular testing using Geno Type Helico DR(Hain Life Science,Germany). This test was performed to detect the presence of H. pylori and to identify point mutations in the genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction from the biopsies,multiplex amplification,and reverse hybridization. RESULTS Clarithromycin resistance was found in 83(16.9%) patients,and fluoroquinolone resistance was found in 66(13.5%) patients. There was no statistical difference in resistance to either clarithromycin or fluoroquinolones(P = 0.55 and P = 0.06,respectively) among the different regions of Brazil. Dual resistance to clarithromycin and fluoroquinolones was found in 4.3%(21/490) of patients. The A2147 G mutation was present in 90.4%(75/83),A2146 G in 16.9%(14/83) and A2146 C in 3.6%(3/83) of clarithromycin-resistant patients. In 10.8%(9/83) of clarithromycin-resistant samples,more than 01 mutation in the 23 S r RNA gene was noticed. In fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. D91 N mutation was observed in 34.8%(23/66),D91 G in 18.1%(12/66),N87 K in 16.6%(11/66) and D91 Y in 13.6%(9/66) of cases. Among fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. CONCLUSION The H. pylori clarithromycin resistance rate in Brazil is at the borderline(15%-20%) for applying the standard triple therapy. The fluoroquinolone resistance rate(13.5%) is equally concerning.

Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

Objective：Duchenne muscular dystrophy （DMD） and Becker muscular dystrophy （BMD） are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification （MLPA） effects of detection of gene mutations. Methods： Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspi- cion of DMD or BMD were tested by MLPA and multiplex PCR. Results ： The mean DQs for all peak of 20 control male samples was 1.02 （range from 0.83 to 1.21） by MLPA. Deletions or duplications were iden- tified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions： dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions： MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detec- tion of DMD and BMD.

Electrochemical and Theoretical Studies of 1-Alkyl-2-substituted Benzimidazoles as Corrosion Inhibitors for Carbon Steel Surface in HCl Medium

The inhibition efficiencies of newly synthesized four 1-alkyl-2-substituted benzimidazole compounds（a^d） have been studied for the corrosion of carbon steel in 1.0 M HCl by using potentiodynamic polarization measurement. The four inhibitors act as mixed-type inhibitors,which mainly inhibit cathodes. The inhibition efficiency of these compounds enhanced when the concentration of the inhibitors increased. A theoretical study of the corrosion inhibition efficiency of these compounds was carried out by using the B3 LYP level with the 6-31＋G＊ basis set. Considering the solvent effect,the IEFPCM model was selected. Furthermore,the adsorption energies of the inhibitors with the iron（001） surface were studied by using molecular dynamic（MD） simulations. The theoretical results show that these inhibitors all exhibit several adsorption active-centers. Meanwhile,the MD simulations indicate that the adsorption occurs mostly through benzene ring and the lone pair electrons of the nitro atoms. These results demonstrated that the theoretical studies and MD simulations are reliable and promising methods for analyzing the inhibition efficiency of organic inhibitors.

Pseudopotential Density-Functional Calculations for Structures of Small CarbonClusters CN （N=2-8）

We introduce a first-principles density-functional theory,i.e.the finite-difference pseudopotential density- functional theory in real space and the Langevin molecular dynamics annealing technique,to the descriptions of structures and some properties of small carbon clusters(C_N,N=2～8).It is shown that the odd-numbered clusters have linear structures and most of the even-numbered clusters prefer cyclic structures.

Microalgal bioinoculants for sustainable agriculture and their interactions with soil biotic and abiotic components:A review

Modern agricultural practices have posed a detrimental impact on the environment due to their intensive use to meet the food demands of an ever-increasing population.In this context,microalgal bioinoculants,specifically cyanobacteria and green microalgae,have emerged as sustainable options for agricultural practices to improve soil organic carbon,nutrient availability,microbial quality,and plant productivity.An overview of current and future perspectives on the use of microalgal bioinoculants in agriculture practices is presented in this review,along with a discussion of their interactions with soil biotic and abiotic factors that affect soil fertility,plant health,and crop productivity.The benefits of microalgal bioinoculants include releasing agronomically important metabolites(exopolymers and phytohormones)as well as solubilizing soil nutrients.Furthermore,they function as biocontrol agents against soil-borne pathogens and facilitate the establishment of rhizosphere communities of agricultural importance.So far,very few studies have explored the basic mechanisms by which microalgal bioinoculants interact with soil biotic and abiotic factors.In recent years,advanced molecular techniques have contributed to a better understanding of these interactions.

Molecularly imprinted polymers for highly sensitive detection of morphine using surface plasmon resonance spectroscopy

Molecular imprinting technology is applied in surface plasmon resonance spectroscopy for highly sensitive and selective detection of morphine（MO）.As SPR-based sensor of MO,the preparation of molecular imprinted polymer is as follows： methacryhc acids（MAA）,ethylene glycol dimethacrylate（EGDMA）,azodiisobutyronitrile（AIBN） were used as functional monomer,cross-linker and initiator,respectively.The experiment results showed that morphine imprinted polymer had the performance of high sensitivity and specificity,i.e.the relative signal of SPR response was proportional to the concentration of morphine in acetonitrile in the range of 10^（-9）mol/L to 10^（-6）mol/L（1 ppb-1 ppm） with LOD of 10^（-10）mol/L,and MO was distinguished from its analogs,such as codeine.

Predicting global numbers of teleomorphic ascomycetes

Sexual reproduction is the basic way to form high genetic diversity and it is beneficial in evolution and speciation of fungi.The global diversity of teleomorphic species in Ascomycota has not been estimated.This paper estimates the species number for sexual ascomycetes based on five different estimation approaches,viz.by numbers of described fungi,by fungus:substrate ratio,by ecological distribution,by meta-DNA barcoding or culture-independent studies and by previous estimates of species in Ascomycota.The assumptions were made with the currently most accepted,“2.2–3.8 million”species estimate and results of previous studies concluding that 90%of the described ascomycetes reproduce sexually.The Catalogue of Life,Species Fungorum and published research were used for data procurement.The average value of teleomorphic species in Ascomycota from all methods is 1.86 million,ranging from 1.37 to 2.56 million.However,only around 83,000 teleomorphic species have been described in Ascomycota and deposited in data repositories.The ratio between described teleomorphic ascomycetes to predicted teleomorphic ascomycetes is 1:22.Therefore,where are the undiscovered teleomorphic ascomycetes?The undescribed species are no doubt to be found in biodiversity hot spots,poorly-studied areas and species complexes.Other poorly studied niches include extremophiles,lichenicolous fungi,human pathogens,marine fungi,and fungicolous fungi.Undescribed species are present in unexamined collections in specimen repositories or incompletely described earlier spe-cies.Nomenclatural issues,such as the use of separate names for teleomorph and anamorphs,synonyms,conspecific names,illegitimate and invalid names also affect the number of described species.Interspecies introgression results in new species,while species numbers are reduced by extinctions.

Patulin Imprinted Nanoparticles Decorated Surface Plasmon Resonance Chips for Patulin Detection

In this study,the patulin imprinted and the non-imprinted nanoparticles are synthesized by the two-phase mini emulsion polymerization method and characterized by zeta-size analysis,Fourier transform infrared spectroscopy,and scanning electron microscopy.Afterwards,the patulin imprinted and the non-imprinted nanoparticles are attached on the surface of surface plasmon resonance(SPR)chips.The patulin imprinted and the non-imprinted SPR nanosensors are characterized by using atomic force microscope,ellipsometer,and contact angle measurements.Kinetic studies for patulin detection are carried out in the concentration range of 0.5nmol-750nmol.The limit of detection and the limit of quantification values are obtained as 0.011 nmol and 0.036 nmol,respectively.In all kinetic analysis,the response time is 13 min for equilibration,adsorption,and desorption cycles.The selectivity studies of the patulin imprinted and the non-imprinted SPR nanosensors are determined in the presence of ochratoxin A and aflatoxin Bl.In order to demonstrate the applicability,validation studies of the patulin imprinted SPR nanosensor are performed by liquid chromatography-tandem mass spectrometry(LC-MS).

Influences and mechanisms of nanofullerene on the horizontal transfer of plasmid-encoded antibiotic resistance genes between E.coli strains

The spread and development of antibiotic resistance globally have led to severe public health problems.It has been shown that some non-antibiotic substances can also promote the diffusion and spread of antibiotic resistance genes(ARGs).Nanofullerene(nC60)is a type of nanomaterial widely used around the world,and some studies have discovered both the biological toxicity and environmental toxicity of nC6O .In this study,cellular and molecular biology techniques were employed to investigate the influences of nC6O at sub-minimum inhibitory concentrations(sub-MICs)on the conjugation of ARGs between the E.coli strains.Compared with the control group,nC6O significantly increased the conjugation rates of ARGs by 1.32-10.82 folds within the concentration range of 7.03-1800μg/L.This study further explored the mechanism of this phenomenon,finding that sub-MICs of nC6O could induce the production of reactive oxygen species(ROS),trigger SOSresponse and oxidative stress,affect the expression of outer membrane proteins(OMPs)genes,increase membrane permeability,and thus promote the occurrence of conjugation.This research enriches our understanding of the environmental toxicity of nC6O,raises our risk awareness toward nC6O ,and may promote the more rational employment of nC6O materials.