Description of a New Cobra (Naja Laurenti, 1768; Squamata, Elapidae) from China with Designation of a Neotype for Naja atra.

Taxonomic frameworks for medically important species such as cobras (genus Naja Laurenti, 1768; Squamata, Elapidae) are essential for the medical treatment of snake bites and accurate antivenin development. In this paper, we described the former N. kaouthia populations recorded from China as a new species and designated a neotype for N. atra-based morphological and mitochondrial phylogenetic analysis. The new species N. fuxisp. nov. was morphologically diagnosed from N. kaouthia by (1) regular single narrow crossband present on the middle and posterior parts of the dorsum (3-15, 7.9 ± 2.7, n = 32) and the dorsal surface of the tail (1-6, 4.2 ± 1.1, n = 32) of both adults and juveniles, buff-colored with dark fringes on both edges, vs. South Asian populations (n = 39) and Southeast Asian populations (n = 35) without cross bands, with irregular cross bands or multiple light-colored crossbands pairs, or densely woven lines; (2) small scales between the posterior chin shields, usually three (40%) or two (37%), rarely four (13%), or one (10%) (n = 30) vs. mostly one (81%) and rarely two (19%) (n = 28); (3) ventrals 179-205 (195.4 ± 6.7, n = 33) vs. South Asian populations 179-199 (188.7 ± 5.9, n = 12); Southeast Asian populations 168-186 (177.8 ± 4.9, n = 18). Phylogenetically, the new species forms an independent sister clade to the clade including N. atra, N. kaouthia, N. oxiana and N. sagittifera. Furthermore, the subspecies N. naja polyocellata should be resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis should be resurrected.

Climate Change and Dispersal Ability Jointly Affects the Future Distribution of Crocodile Lizards.

Crocodile lizards (Shinisaurus crocodilurus) are an endangered, 'living fossil' reptile from a monophyletic family and therefore, a high priority for conservation. We constructed climatic models to evaluate the potential impact of climate change on the distribution of crocodile lizards for the period 2000 to 2100 and determined the key environmental factors that affect the dispersal of this endangered species. For the construction of climatic models, we used 985 presence-only data points and 6 predictor variables which showed excellent performance (AUC = 0.974). The three top-ranked factors predicting crocodile lizard distribution were precipitation of the wettest month (bio13, 37.1%), precipitation of the coldest quarter (bio19, 17.9%), and temperature seasonality (bio4, 14.3%). Crocodile lizards were, just as they are now, widely distributed in the north of Guangdong Province in China and Quang Ninh Province in Vietnam at the last glacial maximum (LGM). Since the LGM, there has been an increase in suitable habitats, particularly in east-central Guangxi Province, China. Under future global warming scenarios, the potential habitat for crocodile lizards is expected to decrease significantly in the next 100 years. Under the most optimistic scenario, only 7.35% to 6.54% of suitable habitat will remain, and under the worst climatic scenario, only 8.34% to 0.86% of suitable habitat will remain. Models for no dispersal and limited dispersal showed that all crocodile lizards would lose habitat as temperatures increase. Our work contributes to an increased understanding of the current and future spatial distribution of the species, supporting practical management and conservation plans.

Ancient Demographics Determine the Effectiveness of Genetic Purging in Endangered Lizards.

The purging of deleterious alleles has been hypothesized to mitigate inbreeding depression, but its effectiveness in endangered species remains debatable. To understand how deleterious alleles are purged during population contractions, we analyzed genomes of the endangered Chinese crocodile lizard (Shinisaurus crocodilurus), which is the only surviving species of its family and currently isolated into small populations. Population genomic analyses revealed four genetically distinct conservation units and sharp declines in both effective population size and genetic diversity. By comparing the relative genetic load across populations and conducting genomic simulations, we discovered that seriously deleterious alleles were effectively purged during population contractions in this relict species, although inbreeding generally enhanced the genetic burden. However, despite with the initial purging, our simulations also predicted that seriously deleterious alleles will gradually accumulate under prolonged bottlenecking. Therefore, we emphasize the importance of maintaining a minimum population capacity and increasing the functional genetic diversity in conservation efforts to preserve populations of the crocodile lizard and other endangered species.

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial).

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO-based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA-anthracycline for low-/intermediate-risk patients, or ATRA-ATO-anthracycline versus ATRA-anthracycline-cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of -5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI -0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan-Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA-chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

A new species of the genus Amolops (Anura: Ranidae) from Yunnan, China.

A new species of the genus Amolops, Amolops tuanjieensis sp. nov., is described from Yunnan, China. The new species can be distinguished by the following characters: dorsolateral folds present; dorsal and ventral surfaces smooth; top of head and dorsum brown-red with irregular gray and dark spots; flank green; side of head black, from tip of snout, diffusing posteriorly to axilla, continuing as black streak below edge of dorsolateral fold; SVL 39.5-40.4 mm in males, 56.8-60.7 mm in females; tympanum distinct; supratympanic fold indistinct; vomerine teeth in two oblique rows between choanae, closer to each other than choanae; vocal sacs present; nuptial pads present; outer metatarsal tubercle absent, supernumerary tubercles absent; all fingertips expanded into discs; limbs dorsally brown with dark brown bars and irregular dark brown blotches.

From mainland to islands: colonization history in the tree frog Kurixalus (Anura: Rhacophoridae).

The origin and colonization history of Kurixalus, a genus of small arboreal tree frogs breeding exclusively in shallow swamps, is under disputed. On the basis of comprehensive sampling program, the evolutionary history of Kurixalus is investigated based on 3 mitochondrial genes. Our results indicate that the genus Kurixalus originated in the Asian mainland and subsequently arrived at its current distribution in Borneo, Taiwan, Ryukyu, and Hainan islands by a series of dispersal events. Moreover, the colonization of Taiwan from mainland Asia has occurred 2 times. The initial colonization of Taiwan occurred at 3.46-8.68 Mya (95% highest posterior density), which rejects the hypothesis that Kurixalus probably originated from Taiwan during the early Oligocene and favors the model of Neogene-origin rather than the model of Quaternary-origin for Taiwanese Kurixalus. Kurixalus eiffingeri has dispersed from Taiwan to the Ryukyus once or 2 times pending more data. Both transoceanic dispersal and landbridge dispersal have played a role in the colonization process; the former resulted in the colonization of Taiwan and the Ryukyus and the latter led to the colonization of Borneo and Hainan.

[Inhibitory Effect of NPM Gene Knockdown on Proliferation of Chronic Myeloid Leukemia Cell Line K562 and Its Mechanism].

OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION: Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.

[Effect of Inhibiting NCL Expression on Drug-Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Resistant Cells].

OBJECTIVE: To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. METHODS: K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. RESULTS: The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ＜0.05), the apoptosis of cells increased significantly (P ＜0.05), and cell resistance to adriamycin was down-regulated. CONCLUSION: Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.

Diets Alter the Gut Microbiome of Crocodile Lizards.

The crocodile lizard is a critically endangered reptile, and serious diseases have been found in this species in recent years, especially in captive lizards. Whether these diseases are caused by changes in the gut microbiota and the effect of captivity on disease remains to be determined. Here, we examined the relationship between the gut microbiota and diet and disease by comparing the fecal microbiota of wild lizards with those of sick and healthy lizards in captivity. The gut microbiota in wild crocodile lizards was consistently dominated by Proteobacteria (∼56.4%) and Bacteroidetes (∼19.1%). However, the abundance of Firmicutes (∼2.6%) in the intestine of the wild crocodile lizards was distinctly lower than that in other vertebrates. In addition, the wild samples from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve also had a high abundance of Deinococcus-Thermus while the wild samples from Guangxi Daguishan Crocodile Lizard National Nature Reserve had a high abundance of Tenericutes. The gut microbial community in loach-fed crocodile lizards was significantly different from the gut microbial community in the earthworm-fed and wild lizards. In addition, significant differences in specific bacteria were detected among groups. Notably, in the gut microbiota, the captive lizards fed earthworms resulted in enrichment of Fusobacterium, and the captive lizards fed loaches had higher abundances of Elizabethkingia, Halomonas, Morganella, and Salmonella, all of which are pathogens or opportunistic pathogens in human or other animals. However, there is no sufficient evidence that the gut microbiota contributes to either disease A or disease B. These results provide a reference for the conservation of endangered crocodile lizards and the first insight into the relationship between disease and the gut microbiota in lizards.

A new species of Limnonectes (Amphibia: Anura: Dicroglossidae) from Vietnam.

A new species of Limnonectes is described from northeastern Vietnam based on morphological and molecular differences. Morphologically, the new species is distinguishable from its congeners on the basis of a combination of the following diagnostic characters: Large size (SVL 50.1-68.9 in males, 45.5-63.0 mm in females); males with moderately enlarged head (HL/SVL 0.48), head longer than wide; vomerine teeth present; external vocal sacs absent; rostral length short (RL/SVL 0.16 in males, 0.15 in females); tympanum distinct (TD/ED 0.63 in males, 0.60 in females); dorsal surface of head, body and flanks with flattened tubercles; dorsal surface of tibia possessing small tubercles; supratympanic fold present; dorsolateral fold absent; webbing formula I0-0II0-1/3III0-1/3IV1/2-0V; in life, dorsum yellowish brown with a dark brown marking; throat and chest white with dark brown marking; ventral surface of fore and hind limbs as well as belly white. In phylogenetic analyses, the new species is placed as the sister taxon to Limnonectes fujianensis with strong statistical support in all analyses.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Microsatellite markers for the invasive species Bidens alba (Asteraceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in the invasive species Bidens alba (Asteraceae) to assess its population structure and to facilitate tracking its expansion in China. • METHODS AND RESULTS: Using 454 pyrosequencing, 20 microsatellite primer sets were developed for B. alba. The markers were tested on one population of B. alba (30 individuals) and one population of the closely related B. pilosa (30 individuals) in China. For B. alba, all of the markers were polymorphic, and the number of alleles per locus ranged from three to 32. The expected heterozygosity values were from 0.3787 to 0.9284, and the Shannon-Wiener index was from 0.6796 to 2.8401. • CONCLUSIONS: These markers will be useful for investigating the genetic structure, genetic diversity, and invasion dynamics of B. alba and will also be useful in studies of B. pilosa.

Complete mitochondrial genome of Shinisaurus crocodilurus (Squamata: Shinisaurus) and its genetic relationship with related species.

The 16,585 base pairs mitochondrial genome of Shinisaurus crocodilurus was determined by using PCR amplification and DNA sequencing. To determine the phylogenetic position of S. crocodilurus with related species within Squamata, the phylogenetic tree was reconstructed with the concatenated nucleotide sequences of the 12 heavy-strand-encoded protein genes. Phylogenetic analyses based on maximum parsimony and Bayesian inference methods consistently support that the S. crocodilurus was closely related to the Helodermatidae within a monophyletic Anguimorpha group. And the result here contradicted the monophyly of Varanoidea (Varanidae + Helodermatidae). In addition, the Gekkonidae was found to possess a basal phylogenetic position within squamata and the traditional hypothesis of monophyletic lineages of Iguania and Scleroglossa was not supported in this study.

Efficacy evaluation for depression with somatic symptoms treated by electroacupuncture combined with Fluoxetine.

OBJECTIVE: This study is to investigate the clinical therapeutic effects and safety of treating mild or moderate depression with somatic symptoms with electroacupuncture combined with Fluoxetine. METHODS: 95 cases of mild or moderate depression with somatic symptoms were randomly divided into a Fluoxetine group, and an electroacupuncture plus Fluoxetine group. Hamilton Depression Scale (HAMD) was used for the assessment of clinical therapeutic effects and Treatment Emergent Symptom Scale (TESS) was used for assessment of adverse reactions. RESULTS: The total effective rate was 77.27% in the Fluoxetine group and 78.26% in the electroacupuncture plus Fluoxetine group, showing no statistically significant difference between these two groups (P > 0.05). However, the treatment took effect after two weeks in the electroacupuncture plus Fluoxetine group but after four weeks in Fluoxetine group. During this time, a better therapeutic effect on depression with mild or moderate somatic symptoms was found in the electroacupuncture plus Fluoxetine group, which also had fewer adverse reactions than the Fluoxetine group. CONCLUSION: Electroacupuncture combined with Fluoxetine takes effect faster for relieving the somatic symptoms with fewer adverse reactions. It is worth popularizing clinically.

[Study on electroacupuncture treatment of depression by magnetic resonance imaging].

OBJECTIVE: To explore the changes of metabolites in brain after treatment by analysis on 1H-MRS examination of the hippocampus and prefrontal lobe in the healthy volunteers and the depression patients. METHODS: Seventy-five cases of mild and moderate depression were randomly divided into groups A, B and C, 25 cases in each group. The group A was treated with oral administration of Prozac capsule, 20 mg/d; the group B by electroacupuncture for 30-40 min and the needle was retained for 1 h, once each day, with main points Baihui (GV 20), Yin-tang (GV 29) and adjuvant acupoints selected; the group C by combination of the treatment methods in the groups A and B. They were treated for 6 weeks. Use PROBE-J sequence at the MRI system on Single Voxel of ROI of each lateral of hippocampus and frontal lobe in the depression patients of the 3 groups. Compare the differences of N-acetylasp artate/creatine (NAA/Cr) and choline/creatine (Cho/Cr) between the healthy volunteers and the patients before and after treatment. RESULTS: Before treatment, NAA/Cr in the bilateral hippocampus decreased in the 3 groups as compared with the control group, and after treatment, NAA/Cr in the bilateral hippocampus of the group B and in the right hippocampus of the group C increased compared with that before treatment (P < 0.05), and NAA/Cr in the left hippocampus of the group C significantly increased as compared with that before treatment (P < 0.01). Before treatment, Cho/Cr in the bilateral prefrontal lobe in the 3 groups increased as compared with that in the control group (P < 0.05), after treatment, Cho/Cr in the bilateral prefrontal lobes of the groups A, B and C significantly decreased as compared with that before treatment (P < 0.05, P < 0.01). CONCLUSION: There are differences in contents of metabolites in corresponding parts in bilateral frontal lobes and hippocampus between the depression patient and healthy person.

[Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing].

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture. These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship. Kefir grains can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. The microorganisms in Kefir grains are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. However, the microorganisms that isolate by these methods can not revert to Kefir grains which indicate that there are some other bacteria that are not isolate from it. In this study, PCR-based Denaturing gradient gel electrophoresis(DGGE) and sequence analysis of 16S ribosomal RNA gene (16S rDNA) clone libraries was used for the rapid and accurate identification of microorganisms from Kefir grains. The PCR primers were designed from conserved nucleotide sequences on region V3 of 16S rDNA with GC rich clamp at the 5'-end. PCR was performed using the primers and genomic DNAs of Kefir grains bacteria. The generated region V3 of 16S rDNA fragments were separated by denaturing gel, and the dominant 16S rDNA bands were cloned, sequenced and subjected to an online similarity search. Research has shown that regions V3 of 16S rDNAs have eight evident bands on the DGGE gel. The sequence analysis of these eight bands has indicated that they belong to different four genera, among them three sequences are similar to Sphingobacterium sp. whose similarities with database sequences are over 98%, three sequences are similar to Lactobacillus sp. whose similarities with database sequences are over 96%, the other two sequence are similar to Enterobacter sp., and Acinetobacter sp. whose similarities with database sequences are over 99% respectively. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, because when universal bacterial PCR primers are used, only the dominant microbiota of an ecosystem will be visualized on a DGGE gel, producing complex banding patterns. However, it could visualize the bacterial qualitative compositions and reveal the major species of the Kefir grains. Among them Sphingobacterium can be found in Kefir grains as the predominant flora which is reported for the first time. PCR-based DGGE and sequence analysis of 16S rDNA proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in microecosystem and probiotic products.