Association of bull semen protein estimates and SDS-PAGE profiles on semen freezability

It is vital to identify the ejaculate with good freezability by determining the biochemical makeup of the ejaculate at the pre-freeze stage. The present study targeted to assess the use of the protein estimates and profiles at the pre-freeze stage as markers of freezability in Frieswal populations. Storing the proteins for proteomic studies is always tricky in the case of animal studies, where accessibility to liquid nitrogen is limited. Hence alternative storing approaches need to be optimized. The second part of this study examined the protein concentration and protein profiles of RNALater and frozen stored sperm cells to assess the use of RNALater preservation in sperm proteomic studies. Sperm and seminal plasma protein concentrations were quantified using Bradford assay, and total protein quantities were derived. The seminal plasma and sperm protein profiles were generated with SDS-PAGE. The protein estimates and SDS-PAGE profiles of good and poor freeze-groups were similar. Also, sperm and seminal plasma protein concentration were not correlated with the semen volume and sperm count. Even though the yield was comparatively less, the protein profiles of sperm preserved by RNALater were similar to that of frozen sperms. The present study results indicate that the protein estimates and qualitative profiles of sperm and seminal plasma proteins may not be sufficient to reveal the differences in the proteome of good and poor freezable bulls at the macro level. Hence, the protein estimates and profiles of neat semen may not be helpful for the prediction of freezability at the pre-freeze stage. Secondly, this study indicates that RNALater preservation helps store sperms for proteome analysis studies.

Biochemical characterization with kinetic studies of melanogenic enzyme tyrosinase from white button mushroom, Agaricus bisporus

In Agaricus bisporus, color is a key determinant for marketability and consumer acceptability. However, postharvest browning has become a major concern, affecting the overall economics of the mushroom industry. In button mushrooms, the tyrosinase enzyme (E.C.1.14.18.1) is responsible for the browning reactions by catalyzing the conversion of monophenols and diphenols into quinones which polymerize to form melanin. Thus, the present study focused on the purification and characterization of tyrosinase from A. bisporus. This enzyme was purified with a final yield of 19.71% and 32.05 purification fold. The study of enzymatic activity over a temperature (5-45°C) and pH range (3-10) showed that the optimum temperature was 35°C with pH 7. The kinetic studies revealed that Km values were different for catechol (0.71 mM) and L-dopa (0.87 mM), which indicated a higher affinity of the enzyme for catechol. Inhibition studies showed that cinnamic acid is a non-competitive inhibitor while salicylic acid is a competitive inhibitor of tyrosinase. The molecular weight of the enzyme was found to be 43 kDa and different amide regions were reflected by the FTIR spectra of the enzyme. This study may provide valuable insights into the structure, biochemical properties, and inhibition of tyrosinase enzyme for controlling mushroom browning.

The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera Psychodidae)

Background & objectives: Sand fly saliva contains proteins that modulate the host immune system and it plays an important role in both blood feeding and the outcome of Leishmania infections. The profile of the salivary proteins was examined and analyzed from an endemic focus of zoonotic cutaneous leishmaniasis by wild P. papatasi to find local and suitable antigens as potential proteins for developing Leishmania vaccine alongside the development of a new extraction technique. Methods: Specimens were caught from Bojnord, using funnel and CDC traps. Different methods of protein extraction were employed and a new technique was developed. The proteins were extracted from the salivary glands tissues with a lysis buffer. Purification was performed using RP-HPLC, with a linear gradient protocol from 0-60 % of acetonitrile. PpSP15 was characterized by SDS-PAGE. Results: The concentration of extracted protein content was 0.5 and 0.03 ?g/?l in chemical and physical methods, respectively. PpSP15 was isolated at a weight of 15kDa in 80–85 min of run time. SDS-PAGE was able to characterize PpSP15. The crude extract of the chemical method, revealed 15 separated bands, ranging from 11–100 KDa. Tajima D index was positive. Interpretation & conclusion: PpSP15 was characterized from Iranian specimens; it is a very highly hydrophobic protein of salivary glands among SP15- like proteins. The chemical method of extraction was found to be more effective than physical methods (P < 0.05). For developing a vaccine against leishmaniasis, depending on the location, choosing suitable proteins should be considered and an efficient extraction method should be used.

Mobilization of seed storage proteins is crucial to high vigor in common bean seeds / Mobilização das proteínas de reserva é crucial para o alto vigor em sementes de feijão

Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.

Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L. ) varieties of North-West Pakistan / Caracterização bioquímica para determinação de distâncias genéticas entre diferentes variedades indígenas de grão-de-bico (Cicer arietinum L. ) do noroeste do Paquistão

Abstract Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.

Resumo As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.

Evaluation of flour protein for different bread wheat genotypes / Avaliação da proteína de farinha para diferentes genótipos de trigo de pão

Abstract Six different bread wheat genotypes; two Egyptian commercial varieties (control); Giza-168 and Gemmeiza-11, and four promising lines; L84 and L148, resulted via hybridization and M10 and M34 via radiation mutation program) were rheologically evaluated using extensograph and for protein, analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The radiation mutant M10 and M34 had the highest maximum resistance which is a very good indicator of strong gluten. The amount of gluten content was higher in M10, L148, and M34 compared to the control samples Gz168 and Gm11. Sulfide amino acids (CYS and MET) are slightly higher in M10. The electrophoretic results and amino acid analyzers show that the best technological quality was exhibited by M10. Radiation mutants wheat genotypes have a protein with good characteristics, mainly gluten which is significantly higher compared to control samples. The rheological properties measured as extensograph and gel electrophoresis were much better in irradiated lines M10 and M34.

Resumo Seis diferentes genótipos de trigo de pão, duas variedades comerciais egípcias (controle) - Giza-168 e Gemmeiza-11 - e quatro linhas promissoras - L84 e L148, obtidas via hibridação, e M10 e M34, via programa de mutação por radiação - foram avaliados reologicamente por meio de extensógrafo, enquanto, para proteínas, foram feitas análises utilizando eletroforese em gel de poliacrilamida com dodecilsulfato de sódio (SDS-PAGE). Os mutantes de radiação M10 e M34 apresentaram a maior resistência máxima, o que é um indicador muito bom de glúten forte. A quantidade de glúten foi maior em M10, L148 e M34 em comparação com as amostras de controle Gz168 e Gm11. Os aminoácidos sulfurados (CYS e MET) são um pouco mais altos no M10. Os resultados eletroforéticos e analisadores de aminoácidos mostram que a melhor qualidade tecnológica foi exibida pelo M10. Os genótipos de trigo mutantes da radiação possuem uma proteína com boas características, principalmente o glúten, que é significativamente maior em comparação às amostras do grupo controle. As propriedades reológicas medidas, como extensógrafo e eletroforese em gel, foram muito melhores nas linhas irradiadas M10 e M34.

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration / Efeitos do flunixin meglumine, meloxicam e firocoxib na concentração de proteínas da fase aguda após em equinos após castração

Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p<0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and α1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.(AU)

Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p<0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida α1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.(AU)

Effects of flunixin meglumine, meloxicam, and firocoxib on the acute-phase proteins in horses following standing castration

ABSTRACT: Excessive infection and inflammation are the most common complications associated with castration. The objective of this study was to compare the efficacy of flunixin meglumine (FM), meloxicam (MX), or firocoxib (FX) for inflammation control after castration in horses using acute-phase proteins (APP) as markers of inflammation. Thirty healthy, unbroken, mixed-breed horses (body weight 358.62±45.57kg and age 4.99±2.63 years) were randomly (n=10 animals/group) allocated to receive one of three different post-castration anti-inflammatory medicines: Group 1 (FM 1.1mg/kg bwt, IV, s.i.d for 5 days); Group 2 (MX 0.6mg/kg bwt, IV, s.i.d for 5 days); and Group 3 (FX 0.1mg/kg bwt, IV, s.i.d for 5 days). All horses were castrated in standing position, using the open technique. Serum and peritoneal APP concentrations were measured by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and determined before castration (0), and 3, 5, 24, 48, 72, 120 and 168 hours after castration. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student-Newman-Keuls test (p 0.05). Three animals from the MX group developed hyperthermia (with rectal temperatures of 39.8, 39.3 and 38.9°C on day 4, 5 and 6, respectively) and showed local clinical signs of inflammation (inguinal and excessive scrotal edema) and reluctance to walk, as well as a rigid gait of the hind limbs. The same complications were observed in one FX horse. No complications were observed among the FM animals. The castration resulted in significant changes in serum and peritoneal values of total proteins, ceruloplasmin (Cp), transferrin (Tf), albumin (Alb), haptoglobin (Hp) and 1-acid glycoprotein (Gp) in animals of all experimental groups. However, the animals of the MX and FX groups presented more intense acute phase response compared to the animals of the FM group. Changes in the APP were associated with the surgical trauma of castration, but the differences between groups were associated with the ability of the nonsteroidal anti-inflammatory drug to control the inflammation. In conclusion, and based on the findings of acute phase proteins, flunixin is more efficient to control the magnitude of inflammation following castration as compared to meloxicam and firocoxib.

RESUMO: Infecção e inflamação excessivas são as complicações mais comuns associadas à castração. O objetivo deste estudo foi comparar a eficácia do flunixin meglumine (FM), meloxicam (MX) ou firocoxib (FX) no controle da inflamação após a castração em cavalos usando proteínas da fase aguda (APP) como marcadores de inflamação. Trinta equinos saudáveis (358,62±45,57kg; 4,99±2,63 anos) foram em função dos anti-inflamatórios utilizados após as castrações aleatoriamente (n= 10 animais/grupo) alocados em três diferentes grupos: Grupo 1 (FM 1,1mg/kg de peso, IV, sid por 5 dias); Grupo 2 (MX 0,6mg/kg de peso, IV, s.i.d por 5 dias); e Grupo 3 (FX 0,1mg/kg de peso, IV, s.i.d por 5 dias). Todos os cavalos foram castrados em posição quadrupedal, utilizando a técnica aberta. As concentrações de APP sérica e peritoneal foram separadas por eletroforese em gel de poliacrilamida (PAGE) com dodecil-sulfato de sódio (SDS) e determinadas no momento 0 (antes da castração) e com 3, 5, 24, 48, 72, 120 e 168 horas após a castração. Os resultados foram submetidos à análise de variância pelo programa estatístico SAS e as médias foram comparadas pelo teste de Student-Newman-Keuls (p 0,05). Três animais do grupo MX desenvolveram hipertermia (com temperatura retal de 39,8, 39,3 e 38,9° C nos dias 4, 5 e 6, respectivamente) e mostraram sinais clínicos locais de inflamação (edema inguinal e escrotal excessivo) e relutância em andar, bem como marcha rígida dos membros posteriores. As mesmas complicações foram observadas em um cavalo do FX. Não foram observadas complicações entre os animais do FM. Independente do grupo, a castração resultou em alterações significativas nos valores séricos e peritoneais de proteínas totais, ceruloplasmina (Cp), transferrina (Tf), albumina (Alb), haptoglobina (Hp) e glicoproteína ácida 1 (Gp). No entanto, os animais dos grupos MX e FX apresentaram resposta de fase aguda mais intensa quando comparados aos animais do FM. Alterações na resposta de fase aguda deveram-se ao trauma cirúrgico da castração, mas as diferenças entre os grupos foram associadas à capacidade do anti-inflamatório em controlar a inflamação. Em conclusão, baseado da resposta de fase aguda, o flunixin em comparação com o meloxicam e o firocoxib é mais eficiente no controle da inflamação após a castração em equinos.

Partial purification and characterization of antimicrobial peptide(s) from Mimosa pudica (Mimosaceae)

@#Plants have been a major source of natural products for sustaining human health. The use of the different parts of the plant as infusions, decoctions, extracts, and powders are being employed in the treatment of different diseases in humans, plants, and animals. One property of great significance in terms of therapeutic treatments, especially with the emergence of multi-drug resistant microbes, is the antimicrobial activity. A new promising source of antimicrobials that demonstrate novel mechanisms of therapeutic strategies is low molecular weight peptides. In this study, the antimicrobial activities of Mimosa pudica crude and partially purified peptide extracts against Gram-negative Enterobacter cloacae ATCC 23355 and Enterobacter aerogenes ATCC 13048, and Gram-positive Staphylococcus epidermidis ATCC 12228 using resazurin colorimetric assay and tricine SDS-PAGE bioautography were reported. M. pudica crude and partially purified extracts exhibited antimicrobial activity against all the bacteria tested. Specifically, the peptide that was partially purified from M. pudica with a molecular weight of 5.14 kDa inhibited the growth of Enterobacter cloacae.

CRISPR/Cas9-Mediated Suds3 Knockout Inhibited Mouse Embryonic Stem Cell Proliferation and Mesoderm/Endoderm Differentiation / 中国生物化学与分子生物学报

Embryonic stem cells (ESCs) have the ability to differentiate into various adult cells, and their fate in the process of development and differentiation is determined by the comprehensive regulation of multiple factors such as gene expression, epigenetics, and extracellular signals. Epigenetic regulation, such as DNA methylation, histone acetylation, and methylation, plays an important role in the maintenance of pluripotency and differentiation of ESCs. Suds3 (Sin3 histone deacetylase corepressor complex component SDS3) is one of the important components of Sin3 histone deacetylase complex. It played an important role in embryonic development, cell proliferation, chromosome separation and other biological processes. However, the functions of Suds3 in ESCs, such as its influence on the proliferation, maintenance of pluripotency and differentiation of ESCs, were rarely reported. In this study, we used CRISPR/Cas9 gene editing technology to construct a Suds3 knockout mouse embryonic stem cell line, and combined cell culture, in vitro embryoid body (EB) formation and in vivo teratoma formation, CCK-8 and cell counting experiments to study the function of Suds3 in ESCs. Western blotting results showed that the SUDS3 protein was not expressed, and the Suds3 gene was successfully knocked out. Through the observation of cell morphology and fluorescence quantitative PCR (QRT-PCR) to detect the expression of pluripotency genes, we found that the knockout of Suds3 had no significant effect on the maintenance of pluripotency of ESCs. Embryoid body (EB) formation experiments revealed that on the fourth and sixth days of EB formation, the pluripotency gene expression was not down-regulated as quickly as WT cells but increased in Suds3

Partial characterization of bacteriocin-like compound (BLIS) produced by Burkholderia stagnalis strain K23/3 against Burkholderia pseudomallei

Aims@#Burkholderia pseudomallei, the human pathogen that causes melioidosis, is intrinsically resistant towards a wide range of antibiotics and there have been reports of acquired resistance towards antibiotics used for melioidosis treatments. Antimicrobial peptides (AMP) such as bacteriocins are gaining the interests of researchers as alternative for treating infections caused by multidrug resistant bacteria. In this study, we aimed to identify Burkholderia spp. isolated from soil in Sarawak that possess the potential in inhibiting the growth of B. pseudomallei and to further characterize the antagonistic compound produced.@*Methodology and results@#A total of 50 Burkholderia spp. isolates of environmental origin and two isolates of Ralstonia solanacearum were screened against five clinical isolates of B. pseudomallei using spot-on-lawn assay and flip streak method. Burkholderia stagnalis isolate K23/3 showed clear zones of inhibition (ZOI) in both preliminary tests. Cell-free supernatant (CFS) was obtained from B. stagnalis K23/3 broth culture and was tested via agar well diffusion assay (AWDA). The antagonistic compound secreted at the early log phase of the bacterial growth was shown to be stable in a wide range of temperatures and pH. Treatment with different enzymes revealed that it was sensitive towards proteinase K, suggesting that it is proteinaceous. The bacteriocin-like-substance (BLIS) was subjected to ammonium sulfate precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel was overlaid with indicator B. pseudomallei isolates where the active protein was shown to be less than 7.1 kDa.@*Conclusion, significance and impact of study@#Burkholderia stagnalis isolate K23/3 was able to secrete bacteriocin-like-substance (BLIS) that has the potential in biocontrol of B. pseudomallei in the environment or as potential treatment for melioidosis.

Evaluation of salivary protein patterns among a rural population exposed and non-exposed to arsenic-contaminated drinking water in areas of Tucumán (Argentina): a pilot study

Abstract Arsenic contamination in the environment and groundwater is a major global public health problem. Several researchers suggest that the toxicity of arsenic could be related to oral cancer development, usually resulting from potentially malignant lesions. During pathological processes, salivary proteins suffer modifications, which could lead to the discovery of new biomarkers. Objective To analyze the protein profile in human saliva samples from a rural population exposed to high levels of arsenic in drinking water and its association with potentially malignant lesions. Methodology This observational, analytic and cross-sectional design included 121 patients from the state of Graneros (Tucumán, Argentina). Arsenic concentration in drinking water was determined and, according to the values obtained, individuals were divided into 2 groups: exposed group and non-exposed group. Saliva samples were obtained, and total protein concentration was measured by Bradford method. Finally, Laemmli SDS-polyacrylamide gel electrophoresis was conducted to obtain the protein profile. Results Total protein concentration in saliva was lower in the exposed group than in the non-exposed group. Average areas of 20 and 42 KDa bands were significantly lower in exposed group than non-exposed group. Conclusion Chronic intake of high arsenic concentrations in drinking water produces changes in the salivary protein profile, which is associated with the presence of potentially malignant lesions.

Biological and Oxidative Effect of Ellagic Acid on Saccharomyces cerevisiae: A New Way for Culture Developing

Abstract In this study, the effects of Ellagic acid (EA) on protein expression in yeasts and cellular development were investigated. Four groups were formed. Groups: 1) Control group; yeast only cultivated group; 2) Ellagic Acid (EA) group: EA (10%) given group; 3) Hydrogen peroxide (H2O2) Group: The group given H2O2 (15 mM); 4) EA + H2O2 group: EA (10%) + H2O2 (15 mM) group. After sterilization, EA (10%) and H2O2 (15 mM) were added to the Saccharomyces cerevisiae (S. cerevisiae) cultures and the cultures were grown at 30 °C for 1 hour, 3 hours, 5 hours and 24 hours (overnight). S. cerevisiae cell growth, lipid peroxidation MDA (malondialdehyde) analysis and GSH (glutathione) level were analyzed by spectrophotometer. Total protein changes were determined by SDS-PAGE electrophoresis and measured by the Bradford method. According to the obtained results, compared with the H2O2 group, cell development (1, 3, 5 and 24 hours), GSH level and total protein synthesis (24 hours) were increased with EA, while MDA level (24 hours) decreased. These results show that EA reduces oxidative damage, increases cell growth and it has a protective effect to promote protein synthesis in S. cerevisiae culture.

Extraction, Partial Purification and Characterization of Bromelain from Pineapple (Ananas Comosus) Crown, Core and Peel Waste

Abstract Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.

Effect of ginger-separated moxibustion on fatigue, sleep quality and depression in patients with chronic fatigue syndrome: a randomized controlled trial / 中国针灸

OBJECTIVE@#To observe the effect of ginger-separated moxibustion on fatigue, sleep quality and depression in the patients with chronic fatigue syndrome.@*METHODS@#A total of 62 patients with chronic fatigue syndrome were randomized into an observation group (31 cases, 3 cases dropped off) and a control group (31 cases, 2 cases dropped off). In the control group, the patients had normal diet and proper physical exercise. In the observation group, on the basis of the control group, the ginger-separated moxibustion was added at Zhongwan (CV 12), Shenque (CV 8) and Guanyuan (CV 4), 30 min each time, once every two days, 3 times weekly. Separately, before treatment and after 4 weeks of treatment, the MOS item short form health survey (SF-36), the Pittsburgh sleep quality index (PSQI) scale and the self-rating depression scale (SDS) were adopted to evaluate the degrees of fatigue, sleep quality and depression in the patients of the two groups.@*RESULTS@#In the observation group, the score of each item of SF-36, the score of each item of PSQI and SDS score after treatment were all improved significantly as compared with those before treatment respectively (<0.05, <0.01). In the control group, the scores of overall health, vitality and mental health in SF-36 and the score of sleep time of PSQI after treatment were improved as compared with those before treatment respectively (<0.05). After treatment, the score of each item of SF-36, the scores of sleep quality, sleep time, sleep efficiency and sleep disorders of PSQI, as well as SDS score in the observation group were all better than those in the control group respectively (<0.01, <0.05). The score of SF-36 was relevant to the scores of PSQI and SDS in the patients of chronic fatigue syndrome ( =0.331, <0.05; =-0.706, <0.01). The improvement value of SF-36 score was closely related to the improvement value of SDS score in the observation group ( =-0.657, <0.01).@*CONCLUSION@#The ginger-separated moxibustion effectively relieves fatigue and depression condition and improves sleep quality in the patients with chronic fatigue syndrome. The fatigue condition is relevant with sleep quality and depression condition to a certain extent in the patients.

Quality evaluation and specification research of Cervus and Cucumis polypeptide injection / 中国药科大学学报

@#To investigate the freshness, high molecular weight substances, the determination of polypeptide, haemolysis and agglomeration, biological activity of Cervus and Cucumis polypeptide injection; to provide the direction for improving the quality of products for enterprises; furthermore, to provide reference for the revision of the quality standards of Cervus and Cucumis polypeptide injection. Firstly, we investigated the factors affecting the freshness of the injection, including biogenic amines, aflatoxins, the acid value and peroxide value of the melon seeds. The method of dansyl chloride pre-column derivatization-HPLC was used to determine the content of 8 biogenic amines in Cervus and Cucumis polypeptide injection. The method validation results showed good specificity, precision, linearity and recovery rates, which was suitable for the determination of biogenic amines in Cervus and Cucumis polypeptide injection. The results of sample determination showed that relatively higher concentrations of cadaverine were detected in the products from company B. The results of aflatoxins, acid value and peroxide value showed that the melon seeds from some companies had rancidity, mildew and other problems, indicating that the quality standards of multi-component biochemical drugs containing animal- and plant-derived components should be controlled in terms of freshness. Secondly, the methods for the determination of high molecular weight substances and polypeptides in the quality standard were improved. Tricine-SDS-PAGE electrophoresis was used instead of gel chromatography to determine the high molecular weight substances, which improved the accuracy of determination. The kits were used instead of folin-phenol for the determination of peptide content, which is easy to operate, specific and suitable for high-throughput sample determination. Finally, the haemolysis, agglomeration, and biological activity of Cervus and Cucumis polypeptide injection were studied. The results showed that no haemolysis and agglomeration were found in all samples, and the inhibitory effect of samples on THP-1 proliferation in vitro from different companies was different to some extent. In conclusion, the optimized quality standard is more suitable for the detection of Cervus and Cucumis polypeptide injection, and can lay the foundation for improving the safety of multi-component biochemical drugs.

Transferrina como marcador de maturação pulmonar em cordeiros nascidos a termo ou prematuros / Transferrin as a marker of pulmonary maturation in lambs born at term or premature

O objetivo do estudo foi procurar proteínas de fase aguda que possam indicar sinais de maturação no neonato prematuro, por meio da quantificação sérica delas. Identificou-se a imunoglobulina A, a ceruloplasmina, a haptoglobina, a glicoproteína ácida, a transferrina, a albumina e as imunoglobulinas G de cadeias leve e pesada, pela comparação do perfil dos proteinogramas de cordeiros nascidos a termo com os prematuros submetidos a diferentes protocolos terapêuticos, a fim de estimular a atividade respiratória. Constituíram-se seis grupos: PN (n= 9): nascidos de parto normal; CN (n= 7): nascidos de cesariana em tempo normal de gestação; CP (n= 6): nascidos de cesariana prematura sem nenhum tipo de tratamento; DEX (n= 9): prematuros cujas mães receberam dexametasona pré-parto; SURF (n= 6): prematuros tratados com surfactante; e DEXSURF (n= 6): prematuros tratados com surfactante cujas mães receberam dexametasona pré-parto. As avaliações foram realizadas nos momentos imediatamente após o nascimento (M0), após 24 (M24) e após 48 horas (M48). As amostras foram processadas por meio de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE). A albumina, as imunoglobulinas e a proteína total dos cordeiros tiveram elevação após a ingestão de colostro. Maiores valores séricos de transferrina são referentes a maior período gestacional, podendo essa proteína ser utilizada como marcador de maturação neonatal.(AU)

The aim of this study was to search for acute phase proteins that could indicate signs of maturation in the premature neonate by quantifying them in serum. Immunoglobulin A, ceruloplasmin, haptoglobin, acid glycoprotein, tranferrin, albumin, light and heavy chain immunoglobulin G were quantified, comparing the profile of proteinograms from term to preterm lambs submitted to different protocols that stimulate respiratory activity. Six groups were used: PN (n= 9): born from normal birth; CN (n= 7): born from caesarean section at normal time of gestation; CP (n= 6): born from premature cesarean without any type of treatment; DEX (n= 9) preterm whose mothers received prepartum dexamethasone; SURF (n= 6) preterm treated with surfactant; DEXSURF (n= 6): preterm treated with surfactant whose mothers received prepartum dexamethasone. The evaluations were performed immediately after birth (M 0), after 24 and 48 hours (M 24 and M 48). Samples were processed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Albumin, immunoglobulins, and serum total protein of the lambs were elevated, after colostrum ingestion. Higher serum transferrin values refer to a longer gestational period, and this protein may be used as a marker of neonatal maturation.(AU)

In vitro Analysis of Antimicrobial Susceptibility of Xanthomonas axonopodis pv. punicae and Comparative Study of Protein Profiling under Biotic Stress

Bacterial blight of pomegranate caused by X. axonopodis pv. Punicae (XAP) assumed epidemic form and resulted in economic burden on farmers. In the current study the pathogen infected samples were collected and the isolated XAP was identity and confirmed through the morphological, biochemical characterization and Pathogenicity test. Bacterium was reisolated from infected plant to prove Koch’s postulates. Efficacy of different chemicals and oils were tested by disc diffusion assay and turbidometrically. Bronopol 3000 ppm (25.6±1.6 mm) and Clove oil (18.0±0.7 mm) formed highest zone of inhibition Turbidometri showed the highest O.D. (0.908 nm) by Copper oxy chloride and Neem oil showed maximum inhibition of growth with O.D. (0.842 nm). Biotic stress (pathogen) induced protein response was studies by using SDS-PAGE method after protein extraction from XAP, healthy P. granatum L. and infected P. granatum L. The protein band pattern showed the unique band no. 2 (Mol.Wt.66000 Da) in infected P. granatum L. as compared to the banding pattern of XAP and healthy P. granatum L. The over expressed protein due to biotic stress could be useful as a marker for detection of the disease at the early stage and for control of the diseases after knowing the biochemical significance of the protein.

Quality selection and genetic diversity of Tunisian durum wheat varieties using SSR markers / Seleção de qualidade e diversidade genética de vari edades de trigo duro da tunísia usando marcadores SSR

Our study focuses on the molecular analysis of the genetic diversity within 15 Tunisian durum wheat varieties and the assessment of the efficiency of some available markers to select valuable genotypes for technological proprieties of semolina (i.e. parameters related to SDS-sedimentation, mixing time and breakdown resistance of mixograph, grain protein content and yellow colour). While several markers were validated, others were not informative within the genotypes used. A high level of polymorphic information content (PIC) was detected, with an average of 5.2 polymorph alleles per locus and 0.6 average. Old varieties have high protein content however; modern varieties display strong gluten strength. Our results thus open the opportunity to choose valuable parents on the base of pedigrees, technological properties and genetic distances; and lead us to select efficient markers for the Regional Indigenous Land Strategy (Rils) selection strategy

Nosso estudo enfoca a análise molecular da diversidade genética em 15 variedades de trigo duro tunisiano e a avaliação da eficiência de alguns marcadores disponíveis para selecionar genótipos valiosos para propriedades tecnológicas de semolina (ou seja, parâmetros relacionados à sedimentação sds, tempo de mistura e resistência à degradação do mixógrafo, teor de proteína dos grãos e cor amarela). Enquanto vários marcadores foram validados, outros não foram informativos dentro dos genótipos utilizados. Foi detectado um alto nível de conteúdo de informação polimórfica (pic), com uma média de 5,2 alelos polimórficos por locus e 0,6 média. Variedades antigas têm alto teor de proteína no entanto; variedades modernas exibem forte força de glúten. Nossos resultados abrem, assim, a oportunidade de escolher pais valiosos com base em pedigrees, propriedades tecnológicas e distâncias genéticas; e nos levam a selecionar marcadores eficientes para a estratégia de seleção da estratégia regional de terras indígenas (rils).

Diversidad genética de tres poblaciones de Physalis peruviana a partir del fraccionamiento y patrón electroforético de proteínas de reserva seminal / Genetic diversity in three populations of Physalis peruvianausing fractionation and electrophoretic patterns of seed storage protein

En el presente trabajo, es estudiada la diversidad genética de tres poblaciones atribuidas a ecotipos de aguaymanto, Physalis peruaviana. Las tres poblaciones eran atribuidas a los ecotipos Agroandino (provincia de San Pablo), Celendino (provincia de Celendín) y Cajabamba (provincia de Cajabamba) del departamento de Cajamarca. Se realizó la cuantificación proteica y evaluó el polimorfismo de las proteínas de reserva seminal (SSPs) mediante electroforesis en gel de poliacrilamida denaturante (SDS-PAGE). Además, se identificaron características bioquímicas de las proteínas seminales en esta especie. No se hallaron diferencias entre las tres poblaciones basados en la cuantificación proteica. Las globulinas (82.4%) fueron la fracción mayoritaria seguida por las albuminas (13.9%), glutelinas (3.7%) y prolaminas (0.7%). Sólo las albuminas mostraron polimorfismo, hallándose 21 proteínas entre ~ 6.5 a ~45 kDa y tres perfiles electroforéticos diferentes, los cuales fueron compartidos entre las poblaciones. Se identificaron las leguminas y vicilinas en la fracción globulina. Las glutelinas mostraron proteínas de mismo peso molecular (PM) a las leguminas; y las prolaminas sólo una banda de bajo PM. La población de San Pablo fue completamente homogénea a diferencia de la población de Cajabamba que mostró la mayor diversidad genética seguida de Celendín. No fue posible diferenciar las poblaciones designadas como ecotipos Agroandino, Cajabamba y Celendino basados en el análisis de proteínas seminales.

The genetic diversity of three populations designated as ecotypes of golden berry (Physalis peruaviana) is studied using protein quantification and polymorphism of seed storage proteins (SSPs) by denaturating polyacrylamide gel electrophoresis (SDS-PAGE). As well, biochemical characteristics of seed proteins were identified. The populations were from San Pablo province (Agroandino ecotype), Celendín province (Celendino ecotype) and Cajabamba province (Cajabamba ecotype), all from Cajamarca Department. There was not difference among the three populations based on protein quantification. Globulins (82.4%) were the majority fraction followed for albumins (13.9%), glutelins (3.7%) and prolamins (0.7%). Only albumins showed polymorphism, showing 21 proteins between ~6.5 to ~45 kDa and three different electrophoretic profiles, which were share among the three populations. Legumins and vicilins were identified in globulin fraction. Glutelins showed proteins of same molecular weight (MW) to legumins; and prolamins only a band of low MW. San Pablo province population (Agroandino ecotype) was completely uniform, while Cajabamba population showed higher genetic diversity followed by Celendin population. Our results shows that, based on seed proteins analyses is not possible to distinguish the three populations designated as Agroandino, Cajabamba and Celendino ecotypes.