RESUMEN
Tape-lifting is a non-destructive method employed in the laboratory to recover and collect trace DNA evidence from crime scene exhibits with porous surfaces. The success of tape-lifting is a balance between capturing the biological material and compatibility with downstream DNA extraction processes to ensure efficient release of the tape-lifted material during DNA extraction. In this study, six commercially available low-, regular- and high-tack adhesive tapes were evaluated. The low-tack S183 tape and the highly adhesive S-Hold tape were compared for DNA recovery efficiency from different materials commonly encountered in casework. All tape-lifts were processed using PrepFiler Express™ BTA and AutoMate Express™ Forensic DNA extraction systems, DNA samples quantitated by Quantifiler TRIO, amplified using Powerplex® 21 and VeriFiler™ PLUS (VFP), and analysed on a 3500xl genetic analyser to evaluate the quality of the resultant STR profiles obtained. The more adhesive S-Hold tape recovered comparable or more DNA than the low-tack S183 tape from the majority of materials tested. However, STR profiles obtained from S183 tape-lifts were of markedly higher quality compared to S-Hold tape-lifts. This was most evident for towel, denim and printed chiffon, where S-Hold samples exhibited severe PCR inhibition, with VFP internal quality markers confirming the presence of inhibitors. The findings suggest that strong adhesion is not necessarily beneficial for tape-lifting, as the low tack S183 tape was able to efficiently recover cellular material from the surface of porous substrates commonly encountered in casework, while avoiding the co-transfer of PCR-inhibitory substances from the sampled material.
Asunto(s)
Dermatoglifia del ADN , Repeticiones de Microsatélite , Humanos , Repeticiones de Microsatélite/genética , Manejo de Especímenes/métodos , ADN/genética , Adhesivos , Reacción en Cadena de la PolimerasaRESUMEN
Testicular apoptosis is involved in the regulation of germ cell numbers, allowing optimal sperm production. Apoptosis has been described to occur in response to the absence of hormonal stimulation of the testis. Here we investigate the effect of the physiological lack of gonadotropins from birth using the hypogonadal (homozygous for the mutant allele Gnrh1(hpg)) mouse as a model. We pursued a concerted strategy using microarray analysis and RT-PCR to assess transcript levels, TUNEL to quantify the incidence of apoptosis, and Western blotting to assess the respective contribution of the extrinsic and intrinsic apoptotic pathways. Our results indicate a large increase in apoptosis of both somatic and germ cell compartments in the hpg testis, affecting Sertoli cells as well as germ cells of all ages. We confirmed our observations of Sertoli cell apoptosis using anti-Mullerian inhibiting substance staining and staining for cleaved fodrin alpha. In the somatic compartment, apoptosis is primarily regulated via the membrane receptor (extrinsic) apoptotic pathway, while in the germ cell compartment, regulation occurs via both the mitochondrial (intrinsic) and membrane receptor (extrinsic) apoptotic pathways, the latter potentially in a stage-specific manner. This study is the first report of spermatogonial apoptosis in response to gonadotropin deficiency as well as the first report of Sertoli cell apoptosis in response to gonadotropin deficiency in the mouse.
Asunto(s)
Apoptosis/fisiología , Gonadotropinas/metabolismo , Hipogonadismo/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Gonadotropinas/genética , Masculino , Ratones , Análisis por Matrices de Proteínas , Transducción de Señal , Espermatogénesis/fisiología , Testículo/citología , Testículo/fisiología , Transcripción GenéticaRESUMEN
Mammary gland involution occurs in two distinct phases: an early, reversible phase, involving extensive apoptosis of the secretory alveolar epithelium without major changes in gland architecture, and a later, irreversible phase, involving remodelling of the gland to its pre-pregnancy state. Multiple signalling pathways are known to be important during early involution, however the precise triggers remain elusive. This review summarizes the roles of a number of key pathways (NF-kappaB, PI(3)K, Stat3, and TGFbeta) in the first phase of involution.
Asunto(s)
Apoptosis/fisiología , Lactancia/fisiología , Glándulas Mamarias Animales/fisiología , Animales , Femenino , Glándulas Mamarias Animales/metabolismo , Ratones , Transducción de Señal , DesteteRESUMEN
Naïve T helper cells differentiate into Th1 and Th2 subsets, which have unique cytokine signatures, activators and transcriptional targets. The Th1/Th2 cytokine milieu is a key paradigm in lineage commitment, and IL-4 (Il4), IL-13 (Il13) and Stat6 are important mediators of Th2 development. We show here, for the first time, that this paradigm applies also to mammary epithelial cells, which undergo a switch from Th1 to Th2 cytokine production upon the induction of differentiation. Thus, the Th1 cytokines IL-12 (Il12), interferon gamma (INFgamma; also known as Ifng) and Tnfalpha are downregulated concomitantly with the upregulation of the Th2 cytokines IL-4, IL-13 and IL-5 (Il5) as epithelial cells commit to the luminal lineage. Moreover, we show that Th2 cytokines play a crucial role in mammary gland development in vivo, because differentiation and alveolar morphogenesis are reduced in both Stat6 and IL-4/IL-13 doubly deficient mice during pregnancy. This unexpected discovery demonstrates a role for immune cell cytokines in epithelial cell fate and function, and adds an unexpected tier of complexity to the previously held paradigm that steroid and peptide hormones are the primary regulators of mammary gland development.
Asunto(s)
Células Epiteliales/fisiología , Interleucina-13/fisiología , Interleucina-4/fisiología , Glándulas Mamarias Animales/crecimiento & desarrollo , Factor de Transcripción STAT6/fisiología , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Interleucina-13/genética , Interleucina-4/genética , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Modelos Biológicos , Embarazo , Factor de Transcripción STAT6/genética , Transducción de Señal/fisiologíaRESUMEN
The Nuclear Factor-kappaB (NF-kappaB) family of transcription factors are ubiquitously expressed and control a wide range of cellular responses, including apoptosis, proliferation, differentiation, inflammation and immunity. Here, we investigated the function of the NF-kappaB upstream regulator IkappaB kinase 2/beta (IKK2) in apoptosis regulation in the normal physiological setting of regressing mammary gland. Conditional deletion of the gene encoding IKK2 resulted, surprisingly, in delayed apoptosis and remodelling, and abrogation of caspase 3 cleavage. This failure to induce involution was associated with reduced expression, within 24 hours of involution, of the death receptor (DR) ligand TNF and its receptor TNFR1, which are known NF-kappaB targets. This was associated with elevated levels of active AKT and phosphorylated FOXO3a. Furthermore, we show that expression of TWEAK, another DR ligand, is dramatically downregulated, even in heterozygous IKK2 mammary glands. Unlike other DR ligands, the TWEAK promoter has six consensus FOXO-binding sites, further suggesting that it is differentially regulated. Interestingly, a cleaved form of TWEAK is upregulated during involution. This unexpected function of the IKK2/NF-kappaB pathway as a regulator of TWEAK expression and inducer of apoptosis has significant consequences for future therapeutic approaches for cancer and inflammatory diseases.