Detection of two CSN1S1 variants in Egyptian buffalo

The present study investigates CSN1S1 casein gene polymorphism in Egyptian buffalo. CSN1S1 was analyzed in 17 unrelated Egyptian lactating buffalo. The amplified segment includes the last 43 amino acids of Exon 17 and part of Intron 17. In the present study we report for the first time the presence of 2 variants 178^Ser (TCA) and 178^Leu (TTA) in Egyptian buffalo CSN1S1 gene. The genotypic frequencies in the investigated Egyptian buffalo sample were 0.47, 0.058 and 0.47 for homozygous 178^Ser, for homozygous 178^Leu and heterozygous 178^Leu/Ser, respectively. The 178^Ser and 178^Leu variant frequencies are 0.64 and 0.36, respectively which indicates the superiority of variant 178^Ser in Egyptian buffalo. The allelic frequency in Egyptian buffalo is not much different from the corresponding allelic frequency in Italian buffalo (0.69 and 0.31 for 178^Ser and 178^Leu, respectively) as reported by Chianese et al. [3]. This is not surprising since they both belong to Mediterranean type.     

Interleukin-21 restrains tumor growth and induces a substantial increase in the number of circulating tumor-specific T cells in a murine model of malignant melanoma

New strategies of immunotherapy are currently being evaluated, and the combination of chemo- and immunotherapy has shown promising results. The cytokine interleukin-21 (IL-21) is known to enhance immune function, and in this study we have investigated its ability to boost the efficacy of chemoimmunotherapy—cyclophosphamide and adoptive cell transfer (ACT)—in the B16-OVA/OT-I murine model of malignant melanoma. Subcutaneous B16-OVA tumors were established in C57BL/6J mice 8 days before adoptive transfer of tumor-specific OT-I T cells. In addition to cyclophosphamide and ACT, one group of mice received daily injections of murine IL-21 (mIL-21). Mice treated with mIL-21 had more tumor-specific T cells in the circulation 4 and 7 days following ACT (P = 0.004 and P = 0.002, respectively). Importantly, mIL-21 and ACT controlled tumor growth instantly and more effectively than ACT alone (P = 0.001, day 4)—an effect that persisted up to 5 days after the last mIL-21 injection. We conclude that mIL-21 enhances chemoimmunotherapy: it amplifies the number of tumor-specific T cells in the circulation and also stunts early tumor growth.     

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n = 18) were split into five aliquots for dilution (37 °C; 50 × 10⁶ spermatozoa ml^?1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0 mM GSH. Extended semen was cooled to 4 °C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5 cm above the LN₂ level) for 10 min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4 h post-thawing (37 °C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0 mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p < 0.05) in all experimental extenders containing GSH when compared to the control extender (0 mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n = 2) spermatozoa was higher (p < 0.05) in extender containing 2.0 mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0 mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.     

Identification of Cu/Zn superoxide dismutase in cattle and river buffaloes

All air-living organisms produce superoxide dismutase (SOD) and several antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species. SOD1 gene has been investigated in Egyptian native cattle and buffalos at the level of genomic DNA and cDNA that were extracted from leucocytes. An unexpected band at approximately 370 bp was obtained in cattle genomic DNA and cDNA as well as in buffalo cDNA. SOD1 amplified sequence of native cattle genomic DNA and cDNA showed a 93% alignment. Native cattle genomic DNA SOD1 amplicon shares sequence homology with mRNAs of Bos taurus “similar to superoxide dismutase” (SOD1) sequence of the GeneBank database. It also shares sequence homology with “similar to superoxide dismutase” on B. taurus chromosome BTA13. The results indicate that the genomic DNA of Egyptian native cattle contains SOD1 processed pseudo gene. SOD1 primers amplified three fragments in buffalo genomic DNA which indicates that buffalo genome has different copies of SOD1 due to alternative splicing. It failed to produce the 370 bp fragments found in cattle DNA. The protein analysis revealed no differences between Egyptian native cattle and B. taurus SOD1 mRNA. However, one amino acid, aspartic acid (Asp), in Egyptian native cattle and B. taurus SOD1, is substituted with asparagine (Asn) (D26N) in buffaloes. This amino acid substitution may be due to non-synonymous single nucleotide polymorphisms (nsSNPs). The nsSNPs detected in buffaloes may affect the function of the encoded protein. This study is the first investigation reporting that the resistance of the buffalo to diseases and parasites that afflict cattle may not be acquired but may have a genetic basis.     

Production of interspecies handmade cloned embryos by nuclear transfer of cattle, goat and rat fibroblasts to buffalo (Bubalus bubalis) oocytes

The possibility of producing interspecies handmade cloned (iHMC) embryos by nuclear transfer from donor cells of cattle, goat and rat using buffalo oocytes as recipient cytoplasts was explored. Zona-free buffalo oocytes were enucleated by protrusion cone-guided bisection with a microblade. After electrofusion with somatic cells, reconstructed oocytes were activated by calcimycin A23187, treated with 6-dimethylaminopurine and were cultured in K-RVCL-50^® medium for 8 days. Although the cleavage rate was not significantly different when buffalo, cattle, goat or rat cells were used as donor nuclei (74.6 ± 3.8, 82.8 ± 5.3, 86.0 ± 4.9 and 82.3 ± 3.6%, respectively), the blastocyst rate was significantly higher (P < 0.01) for buffalo (51.4 ± 2.6) than for cattle (3.5 ± 1.0) or the goat (2.2 ± 0.9), whereas none of the embryos crossed the 32-cell stage when rat cells were used. However, the total cell number was similar for buffalo–buffalo (175.0 ± 5.07) and cattle–buffalo embryos (178.0 ± 11.84). Following transfer of 3 buffalo–buffalo embryos each to 6 recipients, 3 were found to be pregnant, though the pregnancies were not carried to full term. These results suggest that interspecies blastocyst stage embryos can be produced by iHMC using buffalo cytoplasts and differentiated somatic cells from cattle and goat and that the source of donor nucleus affects the developmental competence of interspecies embryos.     

Complete nucleotide sequence and organization of the mitochondrial genome of eri-silkworm,Samia cynthia ricini (Lepidoptera: Saturniidae)

Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A + T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221 bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54 bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNA^Gln and ND2 (54 bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A + T-rich region contains an 18 bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea.     

Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I

Cell cycle progression through its regulatory control by changes in intracellular Ca^2 + levels at the G1/S transition mediates cellular proliferation and viability. Ca^2 +/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr¹⁵⁷ and Thr¹⁹⁸ in human cells and at Thr¹⁷⁰ and Thr¹⁹⁷ in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy.     

Targeting the tumor and its associated stroma: One and one can make three in adoptive T cell therapy of solid tumors

Adoptive T cell therapy (ACT) has become a promising immunotherapeutic option for cancer patients. The proof for ACT therapeutic efficacy was first obtained with allogenic T cells and then reproduced with T cells isolated from patients’ tumor samples (i.e. tumor-infiltrating lymphocytes). It is now clear that specificity of ACT products can be educated by genetically engineering T cells with classical T Cell Receptors (TCR) or chimeric antigen receptors (CAR). To date a poor accessibility of the tumor mass and a hostile microenvironment, influenced by genetic and epigenetic instability, mainly limit ACT therapeutic efficacy in the case of solid tumors. Available data indicate that these hurdles might be overcome by combinatorial therapeutic strategies targeting the tumor and its associated stroma. Here we review some of the available dual targeting strategies focusing on given combination of TCR/CAR-redirected T cell products and their association with drugs targeting the tumor-vessel and/or epigenetic modifiers, with the ability to sensitize tumors to T cell recognition. Existing data have proven synergistic effects in combined settings (one and one can indeed make three) and suggest that further benefit might be achieved by additional combinatorial therapeutic approaches (could one + one + one make ten?) in ACT of solid tumor.     

Isoelectric focusing reveals additional casein variants in German sheep breeds

Isoelectric focusing (IEF) was applied for screening milk protein variants in milk samples from altogether 1078 sheep of different breeds, in detail Black Faced Mutton sheep (SKF; n = 57), East Friesian Milk sheep (OMS; n = 254), Gray Horned Heath (GGH; n = 190), Merinoland sheep (MLS; n = 363), Merino Mutton sheep (MMS; n = 88), and Rhön sheep (RHO; n = 126). Besides the known genetic variants of α_s1-casein (CSN1S1) (A, C, D), α_s2-casein (CSN1S2) (A, B), and β-lactoglobulin (LGB) (A, B, C) additional variants could be demonstrated in CSN1S1 (H, I) and CSN1S2 (C, D) and their genetic control confirmed by segregation analyses. CSN1S1*H corresponds to a previously mentioned phenotype “X” occurring in OMS, whereas CSN1S1*I was identified for the first time in GGH. CSN1S2*C appeared in OMS, GGH, MLS, and RHO in low frequencies and CSN1S2*D in MLS. Within LGB all three alleles occurred in Merino breeds while α-lactalbumin (LAA) and κ-CN (CSN3) were monomorph at protein level. The haplotype CSN1S1*C–CSN1S2*A was predominant in five out of six breeds with frequencies between 0.325 and 0.919.     

Use of lincomycin to control respiratory infections in lambs: Effects on health and production

The efficacy of lincomycin to control respiratory infections in lambs was assessed in two trials. In trial I, 72 lambs with active mycoplasmal pneumonia were allocated as follows: lambs in group T2 were treated with lincomycin (5 mg kg⁻¹ bodyweight, intramuscularly) twice 2 days apart, those in group T3 with lincomycin (5 mg kg⁻¹ bodyweight, intramuscularly) thrice with 2-day intervals, those in group O with oxytetracycline (20 mg kg⁻¹ bodyweight, intramuscularly) twice 4 days apart and those in group C were controls. In trial II, 48 25–30-day-old clinically healthy lambs were allocated as follows: lambs in group P2 received two injections of lincomycin (5 mg kg⁻¹ bodyweight, intramuscularly) when 30- and 60-day-old, lambs in group P1/30 received one injection of lincomycin (5 mg kg⁻¹ bodyweight, intramuscularly) when 30-day-old, lambs in group P1/60 received one injection of lincomycin (5 mg kg⁻¹ bodyweight, intramuscularly) when 60-day-old and lambs in group C were controls. In trial I, treatment with lincomycin was associated with improved clinical scores; clinical cure rate 42 days after treatment was 87%, 100%, 87% and 0% for group T2, T3, O and C, respectively (P < 0.001); treated lambs produced 18.5% (T2) or 26.5% (T3) heavier carcass than controls; no lung lesions were seen in group T3 lambs, whilst they were evident in 22% of group T2 or group O lambs and in 72% of control lambs; microorganisms were isolated from lung tissue samples of 5 group C and 1 group O lambs. In trial II, administration of lincomycin was associated with smaller clinical scores; prevalence rate of respiratory disorders at the end of the trial was 17%, 42%, 42% and 58% for group P2, P1/30, P1/60 and C, respectively (P < 0.01); treated lambs were >4.5% heavier than controls; lung lesions were recorded in 1 group P2, 2 group P1/30 and group P1/60 and 5 group C lambs; microorganisms were isolated from 1 group P2, 3 group P1/30, 2 group P1/60 and 5 group C lambs. It is concluded that administration of lincomycin is effective for the treatment and the prevention of mycoplasmal atypical pneumonia in lambs.     

New genetic polymorphisms within ovine β- and αS2-caseins

Casein genes in ruminants are organized in a cluster including α_S1-casein (CSN1S1), β-casein (CSN2), α_S2-casein (CSN1S2), and κ-casein (CSN3). Considering the results obtained in cattle and goat species concerning the influence of genetic polymorphisms on milk composition, quality, and technological properties, research on the polymorphisms of ewe's milk has known a new impulse in the last decade. A total of 54 samples belonging to the Massese dairy breed, to the double pourpose (milk and meat) Garfagnina population and to the Pomarancina and Zerasca meat populations, reared in the Centre of Italy, were analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). New PCR-SSCP patterns were found in both CSN2 and CSN1S2 genes. Sequencing of the samples carrying the new patterns revealed 2 new variants at CSN2 gene. Frequencies of the 2 variants in the samples analysed were 0.18 and 0.02. The less common variant is characterized by a silent mutation in the triplet coding for Gln₁₉₂, whereas in the more frequent one a C to A transversion is responsible for the aminoacid exchange Leu₁₉₆ → Ile₁₉₆. At the CSN1S2 gene only a new variant was found with a frequency of 0.02. The variant is characterized by two linked mutations: a C to G transversion, responsible for the aminoacid change Asn₂₀₀ → Lys₂₀₀ already described at the protein level, and a T to A transversion at the 14th nucleotide of the 16th intron. The ovine caseins deserve a bigger attention that has to be directed to a complete characterization of the described variants and to the understanding of their functional meaning.     

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma

ObjectiveTo analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4⁺ CD25⁺ T cell (Treg) and its implication in patients with renal carcinoma.MethodsSeventy seven patients are included in the study, and divided into two groups: TRAE group (n = 45, receiving TRAE only) and TRAE + cryoablation group (n = 32, receiving cryoablation 2–3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4⁺T, CD8⁺T, and CD4⁺T/CD8⁺T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy.ResultsThe percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65 ± 1.22)% to (3.93 ± 1.16)%, (t = 42.768, P < 0.01), and the percentages of CD4⁺T and CD4⁺T/CD8⁺T increase significantly (P < 0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4⁺T, CD8⁺T and CD4⁺T/CD8⁺T increase slightly although the differences had no statistical significance (P > 0.05). The tumor necrosis rate of TRAE + cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t = 6.784, P < 0.01). The median survival duration of the TRAE + cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P < 0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r = 0.90, P < 0.01) and life time (r = 0.67, P < 0.01).ConclusionThe therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients‘ survival duration.     

Distribution of TYMS,MTHFR, p53 and MDR1 gene polymorphisms in patients with breast cancer treated with neoadjuvant chemotherapy

Purpose To investigate the role of TSER (TYMS), C677T (MTHFR), Arg72Pro (p53) and C3435T (MDR1) gene polymorphisms in breast cancer patients treated with 5-fluorouracil and cyclophosphamide-based neoadjuvant chemotherapy. Results Observed allelic frequencies were: TSER, (2) 0.54 and (3) 0.46; MTHFR C677T, (C) 0.59 and (T) 0.41; p53 Arg72Pro, (Arg) 0.73 and (Pro) 0.27; MDR1 C3435T, (C) 0.52 and (T) 0.48. MTHFR allele T and p53 allele Pro were strongly associated with toxicity due to chemotherapy (odds ratio, 7.1 (95% confidence interval, 1.4–36.1; p = 0.018) and 7.0 (95% confidence interval, 1.2–40.5; p = 0.029), respectively). Conclusion We introduced new data related to the contribution of p53 codon 72 to toxicity due to 5-fluorouracil and cyclophosphamide-based neoadjuvant chemotherapy in patients with breast cancer.     

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis,spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k_cat = 3.9(4) s^? 1) lower than that of wt SmNir (k_cat = 240(50) s^? 1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N^δ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the N^δ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH 6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway.     

Application of seaweeds for the removal of lead from aqueous solution

Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK₁ were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na⁺, K⁺, Mg²⁺ and Ca²⁺) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.     

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 10⁶ M^?1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 10⁶ M^?1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.     

Effects of rest grazing on organic carbon storage in Stipa Baicalensis steppe in Inner Mongolia

In order to evaluate the potential effects of rest grazing on organic carbon storage on the Stipa baicalensis steppe in Inner Mongolia, compared the S. baicalensis steppes after rest grazing for 3 years, 6 years, and 9 years, using potassium dichromate heating method, this study analyzed the organic carbon storage of plant and soil in the steppes among different periods of rest grazing. The results indicated that as the rest grazing years prolonged, the biomass included above-ground parts, litters and underground plant parts(roots) of the plant communities all increased, meanwhile the carbon content of the biomass increased with the rest grazing years prolonged. For the zero rest grazing (RG0) steppe and the steppes after a rest grazing of 3 years (RG3a), 6 years (RG6a), 9 years (RG9a), the carbon storage in above-ground parts of plant communities were 42.60 g C/m², 66.33 g C/m², 83.46 g C/m², 100.29 g C/m² respectively; the carbon storage of litters were 7.85 g C/m², 9.12 g C/m², 9.18 g C/m², 11.54 g C/m² separately; the carbon storage of underground plant parts (0–100 cm) were 281.40 g C/m², 576.38 g C/m², 745.33 g C/m², 1279.61 g C/m² respectively; and the carbon storage in 0–100 cm soil were 22991.14 g C/m², 24687.75 g C/m², 26564.86 g C/m²,33041.55 g C/m². The results suggested that as the rest grazing years prolonged, the organic carbon storage in plant communities and soil increased. The carbon storage of underground plant parts and soil organic carbon mainly concentrated in 0–40 cm soil. After rest grazing for 3 years, 6 years, and 9 years, the increased soil organic carbon were as the 81.14%, 85.84%, and 89.46% of the total increased carbon; From the perspective of carbon sequestration cost, the total cost of RG3a, RG6a and RG9a were 2903.40 RMB/hm², 5806.80 RMB/hm², and 8710.20 RMB/hm². The cost reduced with the extension of rest grazing years, 0.17 RMB/kg C, 0.16 RMB/kg C, 0.09 RMB/kg C for RG3a, RG6a and RG9a respectively. From the growth characteristics of grassland plants, the spring was one of the two avoid grazing periods, timely rest grazing could effectively restore and update grassland vegetation, and was beneficial to the sustainable use of grassland. From the available data, the organic carbon storage of RG9a was the highest, while the cost of carbon sequestration was the lowest. Therefore, spring rest grazing should be encouraged to continue for it was proved to be a very efficient grassland use measures.     

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel

Ca^2 +-activated Cl⁻ currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl⁻ channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13 μM Ca^2 +. However, in the presence of 1.5 μM Ca^2 + (but not in 13 μM Ca^2 +), A9C also induced a strong potentiation of tail currents measured at − 100 mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.     

Effective technological pectinases by Aspergillus carneus NRC1 utilizing the Egyptian orange juice industry scraps

The production of a notable and highly effective pectinase by the local fungal strain Aspergillus carneus NRC1 utilizing the abundant Egyptian orange peels and pulps (OPP) scraps excluded in the orange juice and canning industry was achieved in 5-day submerged fermentation (SMF) cultures, at temperature and pH ranges of 30–55 °C and 5.0–5.5, respectively. Fresh or thawed OPP (6%, w/v) were the most preferable sole carbon source. Pectinase activity was dramatically stimulated by ammonium sulphate as the sole nitrogen source, and at the same time strongly inhibited the production of the other tested enzymes, i.e., cellulases and hemicellulases. The lyophilized enzyme preparation was free from any ochra or aflatoxins. The optimum conditions of this methodology including enzyme and substrate (citrus pectin) concentrations were 40 mg ml^?1 and 7% (w/v), respectively, with pH and temperature of 4.0 and 55 °C, respectively.