Name: a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model
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Description: Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

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The authors declare that they have no competing financial interests.

Em estudos anteriores, a linha de células C1498 foi descrito como um indutor da granulocítica aguda 5, NKT mielomonocítica 6 ou 7 leucemia de células. No entanto, os dados demonstrativos na literatura estavam ausentes ou incompletos. O protocolo aqui apresentado utiliza técnicas diferentes, como a citometria de fluxo, imunofluorescência, a coloração citoquímica MGG e ensaios, para caracterizar as células C1498 em cultura e para determinar a natureza da leucemia que é induzida em ratos depois de serem injectados. Quando fenotipados em células cultivadas in vitro C1498 após imunocoloração e citometria de fluxo foram realizadas análises, observaram-se algumas limitações porque estas células expressaram alguns marcadores da superfície das células hematopoiéticas que foram previamente descritos na literatura 6,7. De acordo com nossos resultados, Labelle et al. Não observar a expressão na superfície celular de madura TCR em células C1498 usando cytomet fluxocoloração ry. No entanto, eles consideram-nos uma linha de células NKT depois de terem detectado CD3ε e TCRVβ8.2 mRNAs 7. Observou-se também a expressão intracelular de cadeias e moléculas TCRVβ CD3ε na maioria das células (&gt; 70%), mas as suas linhagens hematopoiéticas não pode ser determinada porque havia também a expressão intracelular concomitante da molécula de Mac-3. Mieloperoxidase, MGG coloração e avaliações para analisar esterases funcionais usando citoquímica demonstraram que a linhagem de células C1498 teve uma origem mielóide e foi composto por monoblastos e mieloblastos. Estes resultados foram concordantes com a percentagem de Mac-3 + células que foram obtidos na análise de citometria de fluxo de coloração. Embora não quantitativas, estes passos representam experiências principais para ser realizada. Com efeito, eles permanecem, até agora, os melhores métodos existentes para a caracterização da estirpe e diferenciação de células hematopoiéticas fase que não expressam ou fmarcadores fenotípicos específicos ew. Citometria de fluxo coloração foi útil para demonstrar o desenvolvimento de leucemia aguda em ratos congï¿½icas após C1498 células foram injetadas por via intravenosa. As células C1498 CD45.2 + que se infiltrou num sangue periférico e vários órgãos foram isolados, e as suas frequências foram determinados. Quantificação também foi realizado para analisar medular inerente e as células esplénicas após imunofenotipagem. Limitações foram encontrados quando o fenótipo de células C1498 foi examinada em órgãos como expressaram alguns marcadores hematopoiéticas (apenas alguns deles foram B220 +). Para definir a natureza da leucemia aguda observada, coloração May-Grunwald Giemsa e uma análise das actividades de monocítica e esterases granulocítica foram realizadas utilizando medula óssea. Os resultados mostraram que as células C1498 preservaram a sua morfologia e função mieloblástica e monoblástica, revelando o aparecimento de leucemia mielomonocítica. <p class = &quot;jove_content&quot;&gt; Em consideração para as etapas críticas descritas neste protocolo, deve ser dada especial atenção ao pH ao realizar reações citoquímicas e coloração MGG porque erros no pH podem levar a interpretações incorrectas de resultados. Por exemplo, a actividade de esterase butirato α-naftilo é específico para células monocíticas-se apenas com um pH de 6,0 devido granulócitos e linfócitos também pode manchar positivo para este teste a valores de pH mais elevados. A fixação das células não é recomendado antes de executar coloração MGG, e nós mostrou que apenas a fixação CAF apresentou resultados satisfatórios quando se realiza esterases reações citoquímicas usando C1498 células. Para preservar a expressão da molécula de CD115 e sua detecção por citometria de fluxo, todas as amostras de células (por exemplo, sangue, medula óssea e baço) deve ser mantido em gelo durante o procedimento. Se nenhuma coloração é observada em citometria de fluxo e / ou ensaios de imunofluorescência, a referência dos anticorpos, o seu armazenamento recomendas e suas diluições devem ser verificados. As referências especificadas na tabela de materiais / equipamentos foram selecionados para aplicações de citometria de fluxo ou imunofluorescência. Os anticorpos primários / secundários ou os seus conjugados fluoróforos podem ter perdido a sua actividade devido a armazenamento inadequado (por exemplo, a exposição à luz ou calor), diluição incorrecta, extenso de congelamento / descongelamento ou o uso de tampões contaminados. Executar controlos positivos para garantir que eles estão funcionando corretamente. Use de medula óssea de rato ou células derivadas de baço que são conhecidas por expressar as proteínas de interesse. Para evitar o elevado ruído de fundo e a coloração não específica, certifique-se de que as células são lavadas apropriadamente e mantida a elevada humidade (por imunofluorescência) e que os anticorpos são diluídos conforme instruído. Utilizar a mesma concentração e diluição para o anticorpo de controlo de isotipo e o anticorpo primário para determinar com precisão o nível de fundo na amostra. Para as experiências de esterase citoquímica, oOs reagentes podem ser testados usando lâminas de controlo positivas e negativas contendo rato purificada granulocítica esplénica (Ly6G +) e monocítica (+ CD115) células. O procedimento descrito no presente estudo mostrou que muitas das características leucémicas observados em ratinhos após a injecção de células C1498 partilhada características comuns com leucemia mielomonocítica aguda humano 11,12. As células leucémicas invadiram resultou numa redução de maduras e imaturas (progenitores e de precursores de células hematopoiéticas da medula). C1498 células estão presentes em alta frequência (&gt; 20%) no sangue periférico, como são células monocíticas. Hepatomegalia e esplenomegalia foram observadas como resultado da infiltração das células leucémicas, e aumentos significativos nos linfócitos B e células mielóides também foram observados para acompanhar esplenomegalia. Trombocitopenia foi também observada quando os números de plaquetas do sangue foram estimadas usando um analisador de hematologia. Foi show den, utilizando experimentos in vitro, que C1498 células inibir a hematopoiese murino normal, através da secreção de fatores solúveis 13. Em diversos modelos de ratinho de tumor, as células mielóides imaturas (incluindo monocíticas e células granulocíticas) também têm sido mostrados para migrar a partir da medula óssea para o baço, onde eles inibem a activação das células T específicas do tumor e anti-proliferação 14. Assim, a redução nas células hematopoiéticas que foi observada na medula óssea podem ser resultantes de uma deficiência tanto na hematopoiese e / ou da sua emigração. Este último mecanismo pode explicar a presença de monocitose no sangue periférico ou da observação de fracções mielóides no baço alargada. É também concebível que essas células poderiam ter sido derivado da melhoria da hematopoiese esplénica. De fato, sob condições de estado estacionário, foram identificados alguns subconjuntos de células B do baço como precursores de linfócitos B maduros 15. Além disso, em condições inflamatórias, medAs células estaminais e progenitores ullary foram mostrados para mudar para o baço para induzir a produção de monócitos maduros 16. Este protocolo não permite tirar conclusões sobre os mecanismos que estão envolvidos no desenvolvimento de leucemia, e os ensaios funcionais, bem como moleculares adicionais devem ser empregadas para o fazer. No entanto, estes dados incluem informações detalhadas sobre as características clínicas da leucemia mielomonocítica aguda e ajudará os investigadores a avaliar e compreender os efeitos de novos agentes terapêuticos.

A leucemia mielóide aguda (LMA) é caracterizada pela proliferação não controlada de células mielóides hematopoiéticas que são bloqueados em diferentes estádios de maturação. Esta desregulação pode afetar a granulocítica, monocítica, eritrocítica ou vias de diferenciação megaryocytic 1. células AML acumulam na medula óssea, levando a hematopoiese prejudicada, o que resulta em trombocitopenia, linfopenia e anemia. As células leucémicas também invadem o sangue e órgãos não linfóides. O mouse modelo C1498 tem sido usado por décadas como um modelo para leucemia aguda vez que as células cancerosas foram isolados de um rato leucêmica 10 meses de idade C57BL / 6 (H-2 b) do sexo feminino em 1941. A literatura descreve a invasão no sangue , órgãos hematopoiéticos (por exemplo, o baço e os gânglios linfáticos) e órgãos não-hematopoiéticas (por exemplo, o fígado, pulmões, ovários, rins) e por células C1498 altamente proliferativas após terem sido injectadas por meio de um emintravenosa, subcutânea ou via intra-peritoneal em ratos suscetíveis 2-4. No entanto, neste modelo de ratinho foi relatado para induzir quer granulocítica 2,5 ou 6 mielomonocítica leucemia. Mais recentemente, um estudo publicado em 2002 descreveu este tipo de câncer como leucemia de células NKT murino 7. Assim, a literatura diverge sobre a natureza desta linha de células C1498 ea leucemia associada induz em ratinhos. Estas discrepâncias devem-se principalmente à falta de detalhadas e atualizadas informações publicadas sobre as células e que a doença leucêmica em geral, porque muitos estudos foram realizados na década de 1950 - 70. Aqui nós fornecemos um protocolo detalhado para descrever como a caracterização de células C1498 e analisar a natureza da doença leucémica que é induzida pela sua injecção intravenosa em ratinhos. A primeira seção deste protocolo é dedicado a uma descrição de células C1498 que foram cultivadas in vitro. anti fluorescentecorpos dirigidos contra marcadores de superfície e hematopoiéticas intracelulares foram usadas para determinar o seu fenótipo utilizando citometria de fluxo. A presença de mieloperoxidase foi avaliada utilizando microscopia de imunofluorescência, a sua linhagem hematopoiética e diferenciação fase foram avaliados usando citoquímica para avaliar a actividade de esterase, e coloração May-Grunwald Giemsa foi realizada. As células C1498 foram então injectadas em ratinhos, e a doença de leucemia aguda que foi induzido é descrito na segunda parte deste manuscrito. As mesmas técnicas foram utilizadas para determinar as frequências e os fenótipos de células leucémicas e inerentes na medula óssea, sangue periférico, baço e órgãos não-hematopoiéticas (do fígado e pulmões). Este protocolo é altamente reprodutível, e os dados aqui apresentados ajudará os investigadores a avaliar os efeitos de novas estratégias terapêuticas. Este modelo de leucemia do murganho que já foi usado para testar se aproxima de uma imunoterapiand diferentes fármacos quimioterapêuticos do cancro 8,9. A sua eficácia foi avaliada determinando-se a evolução da carga de tumor e as taxas de sobrevivência. Este protocolo pode ser usado para fornecer informações adicionais sobre a distribuição e de subsistência de populações de células hematopoiéticas leucêmicas e outros durante o tratamento.

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

A injecção intravenosa de células C1498 em ratinhos singénicos ou congénicos foi realizada desde 1941. Estas injecções resultar no desenvolvimento de leucemia aguda. No entanto, a natureza da doença não tem sido bem documentada na literatura. Aqui, nós fornecemos um protocolo técnica para a caracterização de células C1498 in vitro e para determinar a natureza da leucemia induzida in vivo. A primeira parte deste procedimento é focada em determinar a linhagem hematopoiética eo estágio de diferenciação de células C1498 cultivadas. Para alcançar este objectivo, de multi-paramétrico de coloração de citometria de fluxo é utilizada para detectar marcadores de células hematopoiéticas. Microscopia de imunofluorescência, e citoquímica uma coloração May-Grunwald Giemsa são então realizados para avaliar a expressão de mieloperoxidase, a actividade de esterases e morfologia celular, respectivamente. A segunda parte deste protocolo é dedicada a descrever a doença leucemia que é induzido emvivo. Este último pode ser alcançado pela determinação das frequências de células leucémicas e inerentes no sangue, órgãos hematopoiéticos (por exemplo, medula óssea e baço) e tecidos não linfóides (por exemplo, o fígado e os pulmões) utilizando coloração específica e citometria de fluxo análises. A natureza da leucemia é então confirmada utilizando a coloração de May-Grunwald Giemsa e coloração por esterases específicos na medula óssea. Aqui, apresentamos os resultados que foram obtidos utilizando este protocolo em camundongos C1498- e PBS-injetados com a mesma idade.

Para caracterizar o modelo de rato C1498, procedeu com duas etapas principais. Em primeiro lugar, as células C1498 foram caracterizados para determinar a sua linhagem hematopoiética e estado de maturação in vitro (Figura 1). Estas células foram então injectadas em ratinhos congénicos, e a natureza da doença leucémica induzidos foi avaliada para determinar características diferentes: a infiltração de células leucémicas, o seu fenótipo, a quantificação das células hematopoiéticas (maduros e progenitoras / precursoras) na medula óssea, as frequências de células C1498 e células hematopoiéticas maduras no sangue e uma avaliação do inchaço órgão (no baço, fígado e pulmões) e composição celular. Para caracterizar os fenótipos de células C1498 in vitro, as células foram marcadas com anticorpos dirigidos contra moléculas que são expressas pelos precursores hematopoiéticos e células maduras (tabela 1), e os resultados foram analisados por meio de fluxo cytometry. As células C1498 foram positivas para a expressão na superfície celular de Mac-1 (CD11b / CD18) (~ 7%), B220 (&gt; 25%), e eles exibiram expressão intracelular de CD3ε, receptores das células T cadeias (TCR) Vp e MAC -3 (Figuras 2A e B). As células foram negativos para os marcadores de superfície celular Ly6G, Ly6C, CD115, CD21 / CD35, CD19, CD3, CD4, CD8, NK1.1, e pan-NK moléculas e para a expressão intracelular de CD4 e CD8 (dados não apresentados) . Eles foram, em seguida, examinadas para os marcadores de células estaminais hematopoiéticas e progenitoras (Tabela 1). Eles também foram negativos para a expressão na superfície celular de CD117, CD34, Sca-1, CD150 e CD16 / 32 (dados não mostrados). Estas células leucémicas foram então testadas para determinar a expressão da adesão, a apresentação de antigénios e moléculas co-estimuladoras. As células expressaram os marcadores de superfície de LFA-1 (CD11a / CD18), CD44, CD31 (PECAM-1), e H-2D e B foram negativas para MHC de classe II, CD80, CD86 e CD274 (dados não mostrados). células C1498 tor conseguinte expresso tanto mielóide (Mac-1, Mac-3) e marcadores linfóides (B220, CD3, TCR). Para melhor caracterizar sua linhagem hematopoiética, expressão mieloperoxidase foi avaliado usando microscopia de imunofluorescência. Todas as células foram positivas para a mieloperoxidase, a qual verificada a sua origem mielóide (Figura 3A). A maioria das células coradas também positivas para esterases α-naftil butirato (Figura 3B, painel da esquerda), e alguns deles corado para os naftol AS-D cloroacetato esterase (setas pretas) (Figura 3B, painel da direita). Os resultados indicam que as células continham misturas de células monocíticas e granulocítica. Após a coloração May-Grunwald Giemsa foi realizada, as células C1498 foram observados para exibir uma morfologia de explosão do tipo com uma proporção nucleo-citoplasmático alta, 3 a 5 nucléolos no núcleo, halo perinuclear, numerosos vacúolos e citoplasma basofílico (Figura 3C ). ºnós, a linha de células C1498 é composto por monoblastos e mieloblastos. As células C1498 (CD45.2 +) foram então injectados intravenosamente em ratinhos CD45.1 +. Os ratinhos sucumbiu 17 a 19 dias depois, as células foram injectadas. Estes ratos foram sacrificados de modo a que o seu tipo de leucemia pode ser analisada antes de morrer da doença. Os ratinhos de controlo, que foram injectados com PBS, foram analisados ​​no mesmo intervalo de tempo para comparação. Os ratinhos injectados com células C1498 exibida infiltração massiva de células C1498 em sua medula óssea, como demonstrado pelo aparecimento de explosão do tipo das células depois de May-Grunwald Giemsa coloração foi realizada (Figura 4A). Eles também conservaram a sua monocítica e fenótipos granulocíticas (Figura 4B e C), demonstrando uma acumulação de células monoblástica mieloblásticas e que é característica de leucemia mielomonocítica aguda. para determine se medulares números de células hematopoiéticas foram menores após células leucêmicas invasão, C1498 células CD45.2 +, B linfocítica, monocítica e populações granulócitos (incluindo progenitores, precursores e células maduras), foram quantificados utilizando coloração de imunofluorescência e fluxo de multi-paramétricos citometria análise. As células leucêmicas representava 16 a 36% das células hematopoiéticas (dados não mostrados). Os outros tipos de células estavam presentes em números significativamente mais baixos nos ratos C1498 injectados do que nos murganhos injectados com PBS (por 5 vezes, em média, para as subpopulações de células B, 4 vezes em média para células granulocíticas e 3 vezes em média para subconjuntos monocíticas) (Figura 5A a C). Uma investigação das frequências de células mononucleares em amostras de sangue leucêmicas e mouse controle mostrou que eles continham uma percentagem comparável de linfócitos (Figura 6A), mas uma maior freqüência de monocíticae células leucémicas. Estas características são representativas de leucemia mielomonocítica aguda 11 (Figura 6B). Entre as outras características de leucemia mielomonocítica aguda 12, os ratinhos injectados com C1498 apresentados com fígados inchados (hepatomegalia), pulmões e baço (esplenomegalia) (Figura 7A). Várias frequências de células CD45.2 + C1498 foram detectados nestes órgãos utilizando coloração de imunofluorescência e a citometria de fluxo de análise (Figura 7B). Como esplenomegalia pode resultar de um elevado número de monócitos infiltrados, que também estimou as proporções de populações esplênicas. Os números de células B no linfocítica, monocítica e fracções de células granulocíticas foram significativamente maiores, por uma média de 2 vezes, 2,5 vezes e 3-vezes, respectivamente, em baços leucémicas que em baços de controlo (Figura 7C). thin-page = &quot;1&quot;&gt; <img alt="figura 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> Figura 1. Representação esquemática do protocolo descrito acima para a caracterização in vitro linhas de células cultivadas em Descrições C1498 e in vivo de leucemia aguda. A linhagem hematopoiética e a fase de diferenciação das células C1498 em cultura de tecido foram determinados pela primeira vez. Células C1498 foram então injectadas em ratinhos congénicos para induzir o desenvolvimento de leucemia aguda. O isolamento da medula óssea, sangue periférico, baço, fígado e tecidos pulmonares foi realizada para determinar as frequências, fenótipos e alterações morfológicas após a infiltração de células C1498. IV: MGG intravenosa:. May-Grunwald Giemsa <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <html> ad / 54270 / 54270fig2.jpg &quot;/&gt; Figura 2. Análise fenotípica de C1498 células após cultura in vitro. Citometria de fluxo representativas gráficos de pontos e os histogramas de superfície celular (A) e intracelular (B) C1498-expressa moléculas que foram associados com a diferenciação de células hematopoiéticas maduras são mostrados. Células C1498 foram colhidas a partir de culturas, lavadas e marcadas utilizando anticorpos fluorescentes que eram específicos para a superfície celular CD11b, CD18 e B220 marcadores ou os respectivos controlos de isotipo. Para a coloração intracelular, as células foram fixadas, permeabilizadas e marcadas utilizando anticorpos dirigidos contra a Mac-3, CD3ε, e um epitopo comum do TCR (T-Cell Receptor) cadeia Vp ou seus controlos de isotipo. As análises foram realizadas usando gating com células vivas. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <html> ntent &quot;fo: manter-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> Figura 3. funcionais e morfológicas Caracterização de células cultivadas C1498. Células C1498 foram colhidas a partir de culturas e centrifugou-se em lâminas de microscopia. (A) Coloração para a expressão da mieloperoxidase foi realizada utilizando imunofluorescência. (B) reacções citoquímicos foram utilizados para analisar a esterase α-naftil butirato (NBE) e naftol AS-D cloroacetato esterase (CAE) atividades em células C1498. As células foram considerados positivos para cada rótulo quando, grandes grânulos citoplasmáticos marrom e vermelho-púrpura, respectivamente, foram observados. (C) May-Grunwald Giemsa (MGG) coloração de C1498 células. Para cada experimento a coloração, a ampliação microscopia objectivo é indicado. Cada imagem é representativa de três experiências separadas.large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> Figura 4. Os ratinhos de Medula Óssea Morfologias em PBS e injectou-C1498. Células da medula óssea foram isoladas a partir de PBS e ratinhos injectados com células C1498 e centrifugadas em lâminas para microscopia. (A) May-Grunwald Giemsa (MGG) coloração. (B ) esterase α-naftil butirato (NBE) e naftol (C) AS-D cloroacetato esterase (CAE) funções foram avaliados utilizando citoquímica. No painel A, a banda (imaturo) ou neutrófilos segmentados (maduros) são menos visíveis na medula óssea dos ratinhos injectados C1498 do que os ratinhos injectados com PBS. O painel B e C indicam que houve uma acumulação de células monocíticas e granulócitos na medula óssea leucémicas em comparação com os números observados na medula óssea de controlo. Todosanálises microscópicas foram realizadas usando uma objetiva de ampliação 100X. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> Figura 5. Os ratinhos Análise Quantitativa de medulares Populações em PBS e injectou-C1498. Células da medula óssea foram isoladas a partir de PBS e ratinhos injectados com células C1498 e estimado após a contagem das células foi realizada. As frequências dos diferentes populações de células foram determinadas após imunocoloração e fluxo de célula fechada ao vivo citometria de análise. (A) Os subconjuntos de células B incluído células CD19 + B220 + em fases de células pró-B para amadurecer linfócitos B (B) células granulócitos no CD3 - CD11b + e + Ly6G linhagens, que incluiu um precursores. nd granulócitos imaturos e maduros (c) Os subconjuntos monócitos foram definidos como CD3 - CD115 + e as células do progenitor incluída a amadurecer estágios de monócitos. n = 7 ratinhos / grupo, e os dados são apresentados como histogramas mostrando as médias ± SEM. *** P &lt;0,0001 e ** p &lt;0,01, teste t de Student não pareado comparando PBS e ratos C1498-injetados. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> Figura Análise 6. Sangue de subpopulações de células mononucleares em PBS e ratos C1498-injetado. Fluxo representativas citometria de gráficos de pontos de percentagens de linfócitos T (A) e B respectivamente, que foram definidas como as células CD3 + e B220 + em PBS e injectou-C1498 célula. ratinhos (B) As frequências de células monocíticas em leucémicas C1498 e controlo (PBS) murganhos foram determinados através da análise de CD115 + Ly6C - CD115 + e Ly6C células elevados. A análise foi realizada por gating células vivas. Para comparar ratos leucêmicas e controle, CD45.2 + foram excluídos C1498 células. <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 7" src="/files/ftp_upload/54270/54270fig7.jpg" /> Figura 7. Estimativa do baço Populações em leucêmicas e Controle de Ratos. (A) Fotografias representativas de fígado, pulmão e baço inchaço em murganhos leucémicos em comparação com ratinhos de controlo. Os baços foram recolhidos e pesados, e os esplenócitos foram contadas seguinte ruptura de tecido. (B) histograma que representa as frequências de células leucémicas em DIFFórgãos erent após imunocoloração foi realizada para células CD45.2 + e os resultados foram analisados por citometria de fluxo. (C) As estimativas de baço B, o número de células granulócitos e monócitos após imunocoloração e citometria de fluxo gating análises foram realizadas para identificar vivo CD19 + B220 + , CD3 - CD11b + Ly6G +, CD3 - CD11b + Ly6C - e CD3 - CD11b + de células de alta Ly6C. As barras de escala mostradas para os pulmões, fígados e baços indicar 1 cm. .. n = 5-8 ratinhos / grupo, e os dados são representados em histogramas como médias ± SEM * p &lt;0,05; **, p = 0,0033 ratinhos, teste t de Student desemparelhado comparando PBS e injectados C1498 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">favor clique aqui para ver uma versão maior desta figura.</a> <table fo:keep-together.within-page="1"><tbody&gt; <tr><td> Tipo celular </td><td> Membrana ou intracelulares Moléculas </td></tr><tr><td></td><td></td></tr><tr><td> Precursores e células maduras </td><td></td></tr><tr><td> células NK </td><td> NK1.1 +, pan-NK + </td></tr><tr><td> células NKT </td><td> NK1.1 +, pan-NK +, TCR vbeta + (8,2), CD3 + </td></tr><tr><td> linfócitos T </td><td> VBeta TCR +, CD3 +, CD4 +, CD8 + </td></tr><tr><td> células B e os linfócitos B precursores </td><td> B220 +, CD19 +, CD21 / 35 + </td></tr><tr><td> precursores de granulócitos e de granulócitos </td><td> Ly6G +, Mac-1 +, CD11b + </td></tr><tr><td> precursores monócitos / macrófagos e monócitos </td><td> CD11b +, Mac-1 +, Mac-3 +, CD21 / 35 +, CD115 +, Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> progenitores </td><td></td></tr><tr><td> progenitores multipotentes </td><td> CD117 + Sca-1+ CD34 + (Lin CD150-) </td></tr><tr><td> progenitores multipotentes preparado linfóides </td><td> CD117hi Sca-1hi CD127 + (Lin) </td></tr><tr><td> progenitores linfóides comuns </td><td> CD117lo Sca-1lo CD127 + (Lin) </td></tr><tr><td> progenitores mielóides comuns </td><td> CD16 / 32lo CD117 + CD34int (Lin Sca-1) </td></tr><tr><td> progenitores de granulócitos-macrófagos </td><td> CD16 / 32hi CD117 + CD34hi (Lin Sca-1) </td></tr><tr><td> progenitores de megacariócitos-eritróide </td><td> CD16 / 32lo CD117 + CD34lo (Lin Sca-1) </td></tr><tr><td></td><td></td></tr><tr><td> Células-tronco hematopoéticas </td><td> CD117 + Sca-1 + CD150 + (Lin- CD34-) </td></tr></tbody></table> Tabela 1. Os marcadores de linhagens de células hematopoiéticas e diferenciação. CD: cluster de diferenciação; Lin: marcadores de células maduras; lo:baixa expressão; oi: alta expressão; int: expressão intermediária; NK: células assassinas naturais; TCR: receptor de células T.

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Este manuscrito fornece um processo técnico que pode ser utilizado para caracterizar culturas de células C1498 in vitro e a leucemia aguda induzida em ratos após a sua injecção. análises fenotípicas e funcionais são realizadas utilizando citometria de fluxo, microscopia de imunofluorescência, citoquímica e coloração May-Grünwald Giemsa.

Um protocolo detalhado para caracterizar a linhagem celular murina C1498 e seu Modelo Associated leucemia mouse

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

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20.0K visualizações.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC).  Este manuscrito fornece um processo técnico que pode ser utilizado para caracterizar culturas de células C1498 in vitro e a leucemia aguda induzida em ratos após a sua injecção. análises fenotípicas e funcionais são realizadas utilizando citometria de fluxo, microscopia de imunofluorescência, citoquímica e coloração May-Grünwald Giemsa. 

Vídeo: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Balmer Series

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit around the nucleus. Importantly, an atom cannot discharge energy while its electrons are in stationary states. An electron can only emit energy by changing energy states. To change energy states, an electron must move from one orbit to another by either absorbing or emitting energy. This change can only occur if the absorbed or emitted energy equals the difference between the two states. Electrons cannot exist between orbits.
The quantum number, n, is used to label the different energy states. The lowest energy state is the ground state, which is n equal to one. The excited states are labeled n equal to 2, 3, 4, and so on. When an electron at the ground state absorbs a photon whose energy equals the difference between the ground and second state, the electron becomes excited and transitions from the ground state to the n= 2 excited state. If the energy of the photon equals the difference between the ground and third state, the electron moves to the n=3 state.
According to Bohr’s model, the potential energy of an electron in the nth level can be calculated using the following equation:
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_1.jpg" alt="Bohr model energy equation \(E_n=-\frac{Rhc}{n^2}\), quantum mechanics concept." data-altupdatedat="1747908711000">
where En is the potential energy, R is the Rydberg constant (1.0974 × 107 m-1), h is Planck's constant (6.62607004 × 10-34 m2·kg/s), and c is the speed of light (~ 3 × 108 m/s). Electrons can also spontaneously return to the ground state or any other lower excited state. When this happens, the excess energy is released in the form of an emitted photon. The energy of the photon is equal to the energy difference between the higher and lower energy states. That energy corresponds to wavelengths of light. Since each type of atom has different energy levels, the light emitted from each transition varies for each atom. For a sample of mixed molecules, the emitted light contains a range of wavelengths in what is called a continuous spectrum. For a sample containing atoms of a single element, the emitted light contains only certain wavelengths, which can be viewed as discrete lines once separated by a prism.
<h4>The Hydrogen Atom</h4>
Looking specifically at the hydrogen atom, the excitation of its electrons requires the absorption of sufficient energy to split the bond in the diatomic molecule H2. Since more energy is used to split the molecule than needed, the electrons in the hydrogen atom absorb the excess energy and are excited to a higher energy level. When the electrons spontaneously return to a lower energy level, light is emitted, which corresponds to the energy difference between the excited level and lower level.
When discussing the emission of energy, the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. The wavelengths of emitted light ultimately depend on the energy difference between the two levels.
In a pure sample of hydrogen gas, the emission spectrum appears as distinct lines of discrete wavelengths that are specific to the element hydrogen. Some of these lines are in the visible range of the electromagnetic spectrum, while some are in the ultraviolet or infrared range.
<h4>The Balmer Series</h4>
The series of visible lines in the hydrogen atom spectrum are named the Balmer series. This series of spectral emission lines occur when the electron transitions from a high-energy level to the lower energy level of n=2. Johann Balmer observed these spectral lines at 410.2 nm, 434.1 nm, 486.1 nm, and 656.3 nm, which correspond to transitions from the n=6, n=5, n=4, and n=3 energy levels to the n=2 level, respectively.
Balmer was able to relate these wavelengths of emitted light using the Balmer formula.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_2.jpg" alt="Wavelength formula λ=C(m²/(m²−n²)); physics equation; diagram for wave analysis." data-altupdatedat="1747908711000">
Here, λ is the observed wavelength, C is a constant (364.50682 nm), n is the lower energy level with a value of 2, and m is the higher energy level, which has a value greater than 3. This observation was then refined by Johannes Rydberg, where R is the Rydberg constant.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_3.jpg" alt="Rydberg formula for spectral lines, equation, physics, spectroscopy calculation." data-altupdatedat="1747908711000">
Remember that this equation describes emitted light, so the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. In the case of the Balmer series, nf is equal to 2. This equation was combined with the Bohr model to calculate the energy needed to move an electron between its initial and final energy levels, ΔE.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_4.jpg" alt="Energy change formula, ΔE=Rhc(1/nf²-1/ni²), spectral transition equation, quantum physics." data-altupdatedat="1747908711000">
Later, other spectral series for the hydrogen atom were discovered. For example, the Lyman series contains emission lines with energies in the ultraviolet region.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

The Hydrogen Atom: Bohr Model and Balmer Series - Concept

Série Balmer

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit ...

Education

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Química

Genetics of Organisms

<h4>Mendelian Genetics</h4>
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this process as changes in the frequency of alleles at individual genetic loci. This simple representation of how evolution occurs dates to Gregor Mendel&#8217;s analysis of trait inheritance patterns in pea plants, first presented in 1865. Mendel determined, using rigorous collection of data, that noticeable traits are controlled by two alleles of each gene (he called them factors), one from each parent. In addition, he concluded that some alleles are dominant to others (deemed recessive). Interestingly, Mendel&#8217;s data was not appreciated until 1900, probably in part because his approach to the study of inheritance was so divergent from his predecessors. Even after his brilliant experiments were rediscovered, it took 30 additional years for Mendelian inheritance to become integrated with evolutionary theory and to gain universal acceptance.
Why was such a seemingly clear and straightforward process dormant for so long and then the center of controversy for decades? One key to the controversy is that we rarely see the clear patterns of inheritance described by Mendel. Animal and plant breeders most often see evidence for what was described as blending inheritance, or offspring whose phenotypes are intermediate between those of their parents, rather than the discrete segregation of traits into well-defined categories, like smooth versus wrinkled peas or red versus white flowers. Mendelian inheritance thus did not align well with practical experience and it took decades to reconcile the two. Additionally, there are now known to be modes of inheritance that, at first glance, appear to violate Mendel&#8217;s laws. Deeper investigations into genetics using molecular biology have effectively characterized these novel modes of inheritance. The new information does not invalidate Mendel&#8217;s findings, however, it simply enhances his model.
<h4>Phenomena that Violate Mendelian Rules</h4>
There are three main phenomena: epistasis, pleiotropy, and sex-linkage that appear to violate the basic dominance and recessivity rules of Mendelian inheritance. Epistasis occurs when two or more genes or alleles interact to affect a phenotype. A function of one gene product may be required to allow another gene to be expressed or to function normally. On the other hand, pleiotropy occurs when one gene controls the expression of multiple phenotypes in an individual. For example, a large proportion of cats with white fur and blue eyes are deaf 1. Pleiotropy is often antagonistic, meaning that the same gene causes beneficial changes in one facet of an individual&#8217;s phenotype while at the same time causing damaging changes in some other aspect of an individual&#8217;s phenotype. Antagonistic pleiotropy is seen as a tradeoff or constraint on evolution, as proposed by George C. Williams. For example, the age specific decline in performance, or senescence, is thought to be a pleiotropic trait controlled by the same genes that increase early-life fecundity2,3.
<h4>Sex Linkage</h4>
Sex linkage is another phenomenon discovered as a counter-example to basic Mendelian inheritance. In many organisms, pairs of sex chromosomes determine gender. In humans and many mammals, XX individuals are female while XY individuals are male. Male offspring inherit an X from their mother and a Y from their father; females inherit an X from their mother and an X from their father. In most species, the ratio of males to females at birth under &#8220;normal&#8221; environmental conditions is 1:1. Y-chromosomes undergo little recombination with X chromosomes during meiosis, due to the fact that, over evolutionary time, they have lost a substantial number of genes. Interestingly, there are only 16 genes shared by the human X and Y chromosomes4.
For X-linked traits (traits encoded by genes on the X chromosome) females can be homozygous (having two copies of the same allele) or heterozygous (having two different alleles). If the trait is recessive, females will only show the phenotype for the trait if they are homozygous. Heterozygous females &#8220;carry&#8221; the recessive allele, but do not express the phenotype of that allele. Statistically speaking, half of her sons will inherit this recessive allele and all will express the phenotype because males have only one X chromosome.
A commonly used example of an X-linked phenotype is hemophilia, a disease that prevents the synthesis of blood clotting proteins. If a female is heterozygous (is a carrier, genotype XH Xh) mates with a normal male (genotype XH Y), 50% of the sons will be affected. If the female was instead homozygous for the trait (genotype Xh Xh ), none of her daughters would be hemophiliacs but 100% of her sons would be. The differential phenotypic ratios seen in sons and daughters appears to violate Mendel&#8217;s laws, yet Mendel&#8217;s first law still applies; each offspring has a 50% chance of inheriting an X-chromosome carrying the hemophilia allele from a heterozygous mother5.
Like humans, the fruit fly Drosophila melanogaster has an XY-sex determination system. D. melanogaster make excellent laboratory organisms because they are easy to keep, breed, and manipulate. Wild-type flies have what is considered normal body morphology and red eyes. Many lines are readily available for purchase, including some with mutations resulting in flies without eyes, with eyes of varying color, or missing wings. These organisms can be used to study or demonstrate both Mendelian and non-Mendelian inheritance patterns. In fact, 1933 Nobel Prize winner, Thomas Hunt Morgan, first noticed that some genes are sex-linked using this system6.
<h4>References</h4>
<ol class="references">
	<li>Strain, George M., &#39; Cat Breeds With Congenital Deafness,&#39; [Online]. Available: https://www.lsu.edu/deafness/catbreeds.htm. [Accessed 29 August 2018].</li>
	<li>W. GC., &#39;Pleiotropy, natural selection, and the evolution of senescence.,&#39; Evolution, vol. 11, p. 398&#8211;411, 1957.</li>
	<li>T. &#38; S. P. S. Flatt, &#39;Integrating Evolutionary and Molecular Genetics of Aging,&#39; Biochimica et Biophysica Acta, vol. 1790, no. 10, p. 951&#8211;962, 2009.</li>
	<li>D. Bachtrog, &#39;Y chromosome evolution: emerging insights into processes of Y chromosome degeneration,&#39; Nature Reviews. Genetics, Vols. 14(2), 113&#8211;124, 2013.</li>
	<li>US National Library of Medicine, &#39;Hemophilia,&#39; Genetics Home Reference, [Online]. Available: https://ghr.nlm.nih.gov/condition/hemophilia. [Accessed 31 August 2018].</li>
	<li>&#39;The Nobel Prize in Physiology or Medicine 1933,&#39; [Online]. Available: https://www.nobelprize.org/prizes/medicine/1933/summary/. [Accessed 31 August 2018].</li>
</ol>

Genetics of Organisms: REF and WEF Fly Genetics - Concept

Genética de Organismos

Mendelian Genetics
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this ...

Biologia

Genética

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

Comportamento animal

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

Ecologia

Hardy-Weinberg and Genetic Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are therefore very difficult to measure. In general, studying phenomena like this requires the use of mathematical models which are built using parameters that can be conveniently measured. These models are then used to make predictions about how changes to the system might affect the outcome. 
For example, changes to the frequency of genetic alleles at individual loci in species are often observed over long time periods, but are not generally observable over short time periods. The use of computer models allows researchers to predict changes within a species&#8217; gene pool using observations that have already been gathered, and allow for simulations of a potentially unlimited number of generations of the population. This would certainly not be possible within a human lifetime.
<h4>The Hardy-Weinberg Equilibrium Principle</h4>
One equation used to model populations is the Hardy-Weinberg equilibrium equation. It was formulated independently in 1908 by both G. H. Hardy and Wilhelm Weinberg1,2. The simple equation describes the expected allele frequency of a population that is not evolving. Because most real-life populations are evolving in response to the forces of natural selection, this formula acts as a useful null hypothesis. It is used to test whether or not different types of selection are, or are not, occurring. If measured allele frequencies differ from those that were predicted using the Hardy-Weinberg equation, then the gene in question is undergoing evolutionary change.
The Hardy-Weinberg equation can be represented as p2 + 2pq + q2 = 1 where p represents the frequency of the dominant allele and q represents the frequency of the recessive allele for the gene in question. Each expression in the equation represents the predicted frequency of one of the three possible diploid genotypes. Homozygous dominant frequency is represented by p2, homozygous recessive by q2, and heterozygous by 2pq. If these frequencies are summed, it adds up to 1, which makes sense since the full population had been divided into the three available categories.
It is also possible to describe the population, or gene pool, in terms of the two alleles, without regard to how they are packaged into diploid individuals. This equation is represented as p + q = 1, that is, the frequencies of the dominant and recessive alleles must add up to 1 within the population. Again, this makes sense, since the model includes just two allele possibilities. Notice that the frequency of genotypes in the population is just the quadratic expansion of the frequency of alleles in the population because (p + q)2 = p2 + 2pq + q2.
The Hardy-Weinberg equation, like most other models, requires a series of assumptions. Usefully, if the real-life data differs from the predicted data, it is possible to hypothesize which of the starting assumptions was false. The assumptions of the Hardy-Weinberg equilibrium equations are: 1) the population is very large, 2) the population is closed, meaning that there are no individuals immigrating into or emigrating out of the population, 3) there are no mutations occurring on the gene in question, 4) individuals within the population are mating at random&#8212;individuals are not choosing their mates, 5) natural selection is not occurring. Again, keep in mind that the Hardy-Weinberg equilibrium equation is a null hypothesis. Frequently, real populations violate one or more of these assumptions. The equation is used to determine if and how evolution is happening.
<h4>Genetic Drift</h4>
In a relatively small population, a condition that violates the first Hardy-Weinberg assumption, it is possible for allele frequencies to have resulted from chance. This phenomenon is referred to as genetic drift. One version of this is referred to as the founder effect. If a small number of individuals move to an isolated location and start a new population, the specific genetics of those particular individuals will shape the future generations. This new small gene pool may have the same allele frequency as the original, but it is also possible, even likely, that it does not. Say the original population included 50% dominant alleles and 50% recessive. In the extreme, if all of the individuals in a migrating founder group are homozygous recessive, then the dominant allele has been lost completely and the recessive is now at 100%. A similar phenomenon, called the bottleneck effect, can occur if a population sustains a large drop in numbers due to a natural disaster, human intervention, or disease.
Importantly, genetic drift represents a type of evolution that is not necessarily adaptive. It does not specifically select for traits that are fit for the environment. Nonetheless, it is an important evolutionary force in shaping populations with a small number of individuals and will cause deviations from Hardy-Weinberg predictions. In populations that are larger, deviations from the predictions are more likely to mean that the population in question is undergoing evolution through natural selection. Under these circumstances, further consideration of the mechanisms causing this evolution can be explored.
Modern applications of the Hardy-Weinberg model include analysis of human and animal populations in terms of how their immune systems relate to their susceptibility to infectious disease3,4. Many research groups are cataloging genes which encode specific immune system molecules, such as CCR5 and the major histocompatibility complex (MHC, known in humans as the human leukocyte antigen (HLA), and correlating this information to epidemiological studies regarding disease and its progression3,5-6. From comparing these two types of data sets, patterns are emerging showing that some individuals are genetically resistant to particular infections while others are more likely to succumb to disease4. Hardy and Weinberg&#8217;s model has been essential to the analysis of this data and has contributed to our understanding of how infections have shaped evolution.
<h4>References</h4>
<ol class="references">
	<li>Hardy, G. H. (Jul 1908). 'Mendelian Proportions in a Mixed Population' (PDF). Science. 28 (706): 49&#8211;50. doi:10.1126/science.28.706.49. ISSN 0036-8075. PMID 17779291.</li>
	<li>Weinberg, W. (1908). '&#220;ber den Nachweis der Vererbung beim Menschen'. Jahreshefte des Vereins f&#252;r vaterl&#228;ndische Naturkunde in W&#252;rttemberg. 64: 368&#8211;382.</li>
	<li>Eguchi S, Matsuura M. Testing the Hardy-Weinberg equilibrium in the HLA system. Biometrics. 1990 Jun;46(2):415-26. PubMed PMID: 2364131.</li>
	<li>Phillips KP, Cable J, Mohammed RS, et al. Immunogenetic novelty confers a selective advantage in host-pathogen coevolution. Proc Natl Acad Sci U S A. 2018;115(7):1552-1557.</li>
	<li>Lim JK, Glass WG, McDermott DH, Murphy PM CCR5: no longer a 'good for nothing' gene--chemokine control of West Nile virus infection. Trends Immunol. 2006 Jul; 27(7):308-12.</li>
	<li><a target="__blank" href="https://hla-net.eu/tools/frequency-estimation/">https://hla-net.eu/tools/frequency-estimation/</a></li>
	<li>Santiago Rodriguez, Tom R. Gaunt and Ian N. M. Day. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology Advance Access published on January 6, 2009, DOI 10.1093/aje/kwn359. http://www.oege.org/software/hwe-mr-calc.html</li>
</ol>
<h4>Further Reading</h4>
Andrews, C. (2010) The Hardy-Weinberg Principle. Nature Education Knowledge 3(10):65 https://www.nature.com/scitable/knowledge/library/the-hardy-weinberg-principle-13235724

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Concept

Hardy-Weinberg e a deriva genética

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are ...

Evolução

Evolutionary Relationships

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.
<h4>Types of Dendrogram</h4>
A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group1.
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor1. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.
DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.
Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database2. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the query sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.
Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, Drosophila melanogaster, or the mouse3. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.
Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the Cftr knockout mouse and another modeling atherosclerosis, called the Apoe knockout3. 
 
<h4>References:</h4>
<ol class="references">
	<li>Annenberg Learner. Online Textbook: Unit 3 Evolution and Phylogenetics. Rediscovering Biology. [Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li>National Center for Biotechnology Information, U.S. National Library of Medicine. BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li>National Human Genome Research Institute. Background on Mouse as a Model Organism. National Human Genome Research Institute. [Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

Relações evolutivas

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Animal Diversity

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular eukaryotes that move, consume organic matter, and reproduce sexually. Although these attributes are shared, species within this kingdom are also extremely diverse. This diversity is due to adaptation of each species to a different niche. The niche of a species includes the area, function, and interrelationship of that species with other biotic and abiotic factors in its environment. Niche specialization through evolutionary adaptation allows species to survive and reproduce effectively in their environment and reduces competition among species within the same habitat1-2.
<h4>Classification</h4>
Diversity in Kingdom Animalia has led to the classification of 36 distinct phyla, based on their evolutionary lineage. Seven of these phyla will be discussed, including Porifera, Cnidaria, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Chordata. The first, phylum Porifera, is the earliest, simplest, most ancestral phylum. It includes multicellular, asymmetrical filter feeding sponges that lack distinct tissue layers. Interestingly, sponges can regrow after being broken apart to the level of a single cell. Next, increasing in complexity, the phylum Cnidaria includes jellyfish and coral. Members of this phylum are radially symmetric and diploblastic (possessing two tissue layers). These tissue layers are called the endoderm, which makes up the inner layer, and the ectoderm, which forms the outer layer. Organisms in phylum Cnidaria contain a simple gut with a single opening through which food enters and waste is excreted. Both Porifera and Cnidaria include organisms that are largely sedentary as adults. However, the remaining phyla have evolved adaptations that allow movement and more sophisticated behavior.
Following Cnidaria, there are two phyla of worms, termed Platyhelminthes and Annelida. Worms from both of these phyla breathe by exchanging gases across their skin. As a result, most worms have a large surface area to volume ratio and live in moist or aquatic environments. The skin of these organisms must remain moist to facilitate effective diffusion of gases. Another common characteristic of worms is their bilateral symmetry. This means that an organism can be symmetrically divided in half along a plane. Bilateral symmetry is seen not only in worms, but also in every phyla from this evolutionary point forward. Such symmetry is important to allow directed movement and to concentrate sensory organs near the anterior tip, or head, where they interact with their surroundings. Worms and later phyla also contain an additional tissue layer called the mesoderm that lies between the ectoderm and endoderm. As a result, these organisms are considered &#8220;triploblastic.&#8221;
Of the two phyla of worms, Platyhelminthes is the more ancient and simple. These include flatworms and trematodes, which may be free-living or parasitic. Like Cnidarians, they possess a simple gut with a single opening. Phylum Annelida is more complex, including earthworms, leaches, and polychaeta. These organisms contain a body cavity, called the coelom, between the gut and body wall. They are also distinct due to the segmentation of their bodies. Division of an organism into segments was an important evolutionary step, allowing adaptation of each segment to specialized forms and functions. Major evolutionary advances in this phyla include the development of anterior segments with appendages specialized for grasping and processing food as well as posterior segments with appendages specialized for locomotion.
The next phylum, Mollusca, contains highly morphologically diverse organisms, including octopuses, snails, slugs, and clams. There are few defining features for this group. However, all mollusks possess a mantle, which acts as a protective cover for the respiratory organs, digestive tract, and reproductive structures. Next, the most diverse group of organisms is phylum Arthropoda. This phyla alone makes up approximately three quarters of all living and extinct species known today, including crustaceans, insects, and arachnids. All members of this phylum have a hard, outer coating made of chitin, called an exoskeleton. This exoskeleton is periodically shed for a new, larger one to take its place. Like Annelida, arthropods are segmented. However, instead of multiple repeating sections, arthropods are broken up into three distinct segments called the head, thorax, and abdomen. Most have specialized sensing structures attached to the head called antennae.
Lastly, phylum Chordata includes all animals with a backbone and a few without. Two defining features that unite this group are the presence of a nerve chord and a notochord. The nerve cord is a major part of the chordate nervous system and the notochord is a cartilaginous structure that runs between the nerve cord and the digestive tract. Phylum Chordata includes tunicates (which lose their nerve cord and notochord after metamorphosis from the larval to adult life stage), fish, amphibians, birds, reptiles, and mammals (including humans)3.
<h4>Form and Function</h4>
The vast diversity in body structure, habitats, and behavior observed across all 36 phyla within Kingdom Animalia is truly astounding. To understand these differences better, it is important to consider the connection between form and function. &#8220;Form&#8221; simply means the shape, size, and substance of a structure or organism while &#8220;function&#8221; stands for the way in which that structure is used. Accordingly, the function of a structure can be predicted based on the form observed, or vice versa. For example, a bird specialized to crack hard nuts would be predicted to have a sturdy, sharp beak while a bird that eats fruit may have a smaller, more rounded beak.
Importantly, the form of an organism is constrained by its physiological requirements. For instance, all animals must consume and digest organic matter and exchange gas with their environment. As a result, any adaptations to an animal&#8217;s form must continue to facilitate these processes efficiently. For example, in smaller animals, diffusion of gases across the skin is possible and often useful. However, as the size of an animal increases, its surface area to volume ratio decreases until diffusion across the skin can no longer provide sufficient gas exchange. Such larger animals have developed complex systems to deal with gas exchange, such as the lungs of a whale or the gills of a fish. As a result, the morphology of gas exchange systems depends on both the size of an organism and its environment.
Similarly, it generally follows with other types of adaptations that form follows function, constrained only by the physiological requirement of the organism. However, there are cases in which form does not follow function. Vestigial structures are those that served a purpose in an organism&#8217;s ancestor but are no longer useful in their current form. Such structures have little to no impact on the fitness of an individual, and are thus not evolutionarily selected for or against within the population. As a result, some structures persist with limited functional capacity. For example, the human appendix, a small pocket at the end of the large intestine, is an evolutionary remnant from our ancestors. While this structure has been hypothesized to store beneficial gut bacteria, removal of the appendix is possible with no deleterious effects.
<h4>Convergent Evolution</h4>
Among adaptations seen in Kingdom Animalia, many have developed multiple times throughout evolutionary history. Consider the wings of a bat and those of a bird as an example. These structures are not a result of evolutionary relatedness, but have instead arisen from convergent evolution4. Convergent evolution occurs when two organisms independently evolve similar traits due to similar environmental pressures or niches. The wings of bats, birds, and insets have all developed independently and are said to be &#8220;convergent traits.&#8221; Clearly, such traits are quite beneficial for the niches in which they arise.
<h4>References</h4>
<ol class="references">
	<li>Hollins, J., et al. (2018). 'A physiological perspective on fisheries-induced evolution.' Evol Appl 11(5): 561-576.</li>
	<li>Leong, M., et al. (2017). 'The Habitats Humans Provide: Factors affecting the diversity and composition of arthropods in houses.' Sci Rep 7(1): 15347.</li>
	<li>Wray, G. A. (2015). 'Molecular clocks and the early evolution of metazoan nervous systems.' Philos Trans R Soc Lond B Biol Sci 370(1684).</li>
	<li>Foll, M., et al. (2014). 'Widespread signals of convergent adaptation to high altitude in Asia and America.' Am J Hum Genet 95(4): 394-407.</li>
</ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Concept

Diversidade Animal

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular ...

Microbial and Fungal Diversity

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this reason, it is important to understand and distinguish between individual species of these groups. As you will recall, biological taxonomists group organisms based on their phylogenetic relatedness. The three domains of life, Bacteria, Archaea, and Eukaryota segregate life into three distinct groupings. While all bacteria fall within the domain Bacteria, fungi are found in the domain Eukaryota (Kingdom Fungi). In addition to Archaea, domain Bacteria exclusively includes single-celled prokaryotes, meaning their cells lack both a nuclear membrane and membrane-bound organelles. In contrast, fungi of domain Eukaryota have membrane-bound organelles and include both unicellular and multicellular organisms.
<h4>Bacteria</h4>
Bacteria are found nearly everywhere on Earth and are highly adapted to the environments in which they grow. As a result, this domain includes a staggering amount of diversity. The impact of these organisms on human health is equally diverse. For example, species like Streptococcus thermophilus can be used to produce fermented foods like yogurt, while spores of the bacteria Bacillus anthracis are responsible for the potentially deadly disease anthrax.
To aid in the identification of such organism, microbiologist have developed a myriad of tests to characterize both individual cells and entire colonies of bacteria1. One of the most common methods for identifying bacteria is by Gram staining. This test uses colored dyes to determine the composition of the bacteria&#8217;s cell wall. Gram-positive bacteria, which bear a thick cell wall made up of a protein called peptidoglycan, retain a dye called crystal violet and thus appear purple. In contrast, gram-negative bacteria have a thinner cell wall without peptidoglycan and do not retain the crystal violet. Instead, these cells appear pink due to a counter stain called safranin. The use of Gram staining often serves as a preliminary step in the identification of a bacterial species. Further classification requires the identification of additional characteristics. This can include the shape of individual bacteria, which may be either cocci (round), spirillum (spiral), or bacilli (rod-shaped). Additionally, the color, shape, sheen, texture, and even smell of bacterial colonies can be used to distinguish between species.
<h4>Fungi</h4>
Fungi of domain Eukaryota are also highly ecologically diverse and can range in size from microscopic yeast to the largest organism on earth, the honey fungus (Armillaria ostoyae). All fungi are heterotrophic, meaning that they must acquire their food from the environment. These organisms also exhibit diversity in their specific activities, niches, and potential effects on human health. For example, some fungi are consumed or used to produce food by humans while others can cause severe or even deadly infections2.
Within kingdom Fungi, two distinct taxonomic divisions exist based on the reproductive strategy of the species. First, Ascomycota, known as the sac fungi, have a reproductive structure called an ascus. Together, many asci form the ascocarp, or &#8220;fruiting body&#8221; of the fungi. These fungi can reproduce sexually or asexually by budding. Ascomycota consist of some economically important species like the yeast used in brewing beer and baking bread, and some species popular in cuisine, such as morels and truffles. The second group, Basidiomycota, includes organisms commonly known as &#8220;club fungi&#8221; due to the presence of a large club-shaped reproductive structure called the basidium. In contrast to Ascomycota, most Basidomucota reproduce exclusively by sexual reproduction. This occurs when two haploid mycelia join to form a basidiocarp. On a button mushroom, this is the cap structure. On the underside of the basidiocarp, reproductive structures called gills lined with structures called basidia produce haploid nuclei that eventually form basidiospores. Basidiospores are then dispersed by the wind. This taxonomic division includes many commonly eaten button mushrooms, as well as wild puff mushrooms.
<h4>Importance of Microbiology</h4>
Overall, it is clear that the diverse functions and properties of microorganisms make their identification extremely important for human health and well-being. While a vast number of species of fungi and bacteria have been identified to date, it is estimated that millions more have yet to be identified and classified. In addition, human disciplines that explore Fungi and Bacteria are as diverse as the organisms themselves and will undoubtedly continue to grow. The relevance of microorganisms to the medical fields is more prevalent now then ever, as multi-antibiotic resistant species of infectious bacteria threaten human health. Understanding the structure and biological characteristics of infectious and non-infectious species provides key insights necessary for the creation of new antibiotic treatments and preventative strategies3-4.
Microbiology is also critical to the food industry. Numerous products, including yogurt, cheese, kombucha, bread, and alcohol are made using specific categories or species of microbes. At the same time, food scientists focus on engineering chemical compounds and preservatives that will reduce the growth of unwanted microbes on food. In all these processes, an understanding of how microbes survive, reproduce, and affect their environment is necessary.
<h4>References </h4>
<ol class="references">
	<li>Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi, &#8220;Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance,&#8221; BioMed Research International, vol. 2014, Article ID 725801, 2 pages, 2014. https://doi.org/10.1155/2014/725801.</li>
	<li>Treseder, K. K. and J. T. Lennon (2015). 'Fungal traits that drive ecosystem dynamics on land.' Microbiol Mol Biol Rev 79(2): 243-262.</li>
	<li>Arvanitis, M. and E. Mylonakis (2015). 'Fungal-bacterial interactions and their relevance in health.' Cell Microbiol 17(10): 1442-1446.</li>
	<li>Aarestrup, F. M., et al. (2008). 'Resistance in bacteria of the food chain: epidemiology and control strategies.' Expert Rev Anti Infect Ther 6(5): 733-750.</li>
</ol>

Microbial Colony and Fungal Diversity - Concept

Diversidade microbiana e fúngica

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

Divisão Celular

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

Processos Celulares

Lab Manual Sub Articles

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Bacterial Transformation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">First, ensure that each student or student group has a filled ice bucket.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, set up a tube rack for each work station.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set out two LB agar plates per group and two LB agar ampicillin plates per group.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Ensure the students have the appropriate pipettes.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">And finally, set a water bath to 42 &#176;C and a temperature-controlled shaker to 37 &#176;C.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Prep

Transformação bacteriana

Bacterial Transformation
ExpandFirst, ensure that each student or student group has a filled ice bucket.
Then, set up a tube rack for each work station.
...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Cheek Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Immediately before the experiment, wash and peel onion bulbs for the class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Remove the entire brown outer skin and cut the onion in half with a knife. Pull apart the layers of the onion. The thin, nearly transparent film layers within the onion will be used by the students.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the onion film into a Petri dish and then place this into the refrigerator until the students arrive.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Bring out the microscopes and set them on the lab bench.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the microscopes to the lowest magnification and plug them into an electrical outlet.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Clean the lenses with lens paper or laboratory wipes and test the knobs to make sure the lenses focus properly.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Make sure that the stage can be moved.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Prep

Estrutura Celular

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...