RESUMEN
BACKGROUND/AIM: Coronavirus-19 (COVID-19) pandemic had a huge impact on medical resource allocation. While it is clear that the surgery refusal rate of patients with breast cancer (BC) was higher during the pandemic, long-term effect of COVID-19 pandemic on hospital admission in the post-pandemic period has not been fully evaluated. This study aimed to estimate how patients' behavior changed following the pandemic and whether the cross-infection risk is still influencing patients' decision-making process. PATIENTS AND METHODS: Between the 16th of January and 18th of March 2020, between 19th of March 2020 and the 20th of March 2020, and between 19th of March 2023 and the 20th of March 2023, 266 patients were enrolled and divided into PRE-COVID-19, COVID-19, and POST-COVID-19 groups, respectively. A total of 137 patients with a suspected breast lesion (SBL) were divided into 3 groups: PRE-COVID-19-SBL, COVID-19-SBL, and POST-COVID-19-SBL groups. In addition, 129 BC patients were divided into PRE-COVID-19-BC, COVID-19-BC and POST-COVID-19-BC groups. Patient characteristics including age, marital status, SBL/BC diameter, personal and family history of BC, clinical stage and molecular subtype were recorded. Procedure refusal (PR) and Surgical refusal (SR) were also recorded with their reason. RESULTS: BC and SBL analysis showed no difference in pre-treatment characteristics (p>0.05). While higher rate of PR and SR rates were reported in COVID-19-SBL and COVID-19-BC groups when compared with PRE-COVID-19 (p=0.003, p=0.013, respectively) and POST-COVID-19 (p=0.005, p=0.004, respectively) groups, no statistical difference was found between PRE-COVID-19 and POST-COVID-19 subanalysis. CONCLUSION: Thanks to preventive measures, COVID-19 does not currently seem to affect the decision-making process of patients with BC.
Asunto(s)
Neoplasias de la Mama , COVID-19 , Humanos , Femenino , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/patología , COVID-19/epidemiología , Pandemias , SARS-CoV-2 , Ansiedad , MiedoRESUMEN
BACKGROUND/AIM: Despite an aging population, there is no consensus regarding ductal carcinoma in situ (DCIS) treatment for elderly women. Breast surgery can be well tolerated even in elderly patients. The aim of this study is to evaluate the surgical management of DCIS in elderly patients. PATIENTS AND METHODS: We retrospectively analyzed patients with DCIS from 2016 to 2022 at our Breast Unit and divided our population according to age. RESULTS: Out of 231 patients with DCIS, 45 (19.5%) were elderly. The Charlson comorbidity index and American Society of Anesthesiology (ASA) score was significantly higher in the elderly (p<0.001 for both). Among the elderly, 10 (22.2%) patients received upstaging diagnoses, versus 18 (9.7%) in the control (p=0.048). Twelve (26.7%) of the elderly patients underwent sentinel lymph node biopsy, versus 93 (50%) in the control group (p=0.005). No difference was reported between groups in terms of breast conserving surgeries performed. A higher incidence of surgeries performed using local anesthesia was reported in the elderly group (p=0.041). Thirty-day surgical complications, according to Clavien-Dindo, did not show significant differences. CONCLUSION: Despite higher comorbidity and ASA score, breast surgery is safe and feasible in elderly patients. Due to the higher risk of upstaging to invasive ductal carcinoma, surgery should be performed but sentinel lymph node biopsy should be omitted, owing to the low risk of lymph node metastasis and lower use of adjuvant treatments.
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Neoplasias de la Mama , Carcinoma Ductal de Mama , Carcinoma Intraductal no Infiltrante , Humanos , Femenino , Anciano , Carcinoma Intraductal no Infiltrante/cirugía , Carcinoma Intraductal no Infiltrante/patología , Estudios Retrospectivos , Biopsia del Ganglio Linfático Centinela , Metástasis Linfática , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/patologíaRESUMEN
Siblings are often cited as a source of support throughout life, but they have not been included in studies of sexual violence disclosure. Survivors of sexual violence often delay or do not disclose their sexual violence. The current study investigated if and why survivors of sexual violence decided to disclose to their sibling. This qualitative investigation included 10 female participants, of which six had disclosed their abuse to their sibling. The participants ranged in age from 20 to 58, five identified as Hispanic, one as Hispanic and White three identified as White, and one identified as Middle Eastern. Thematic analysis was used to examine the data. Three major themes were identified that affected sibling disclosure decisions: sibling dynamics, perceived and real reactions to disclosure, and words of advice. The findings from this study demonstrate the continued need to investigate siblings and their role in disclosure of sexual violence. Practitioners may also want to examine the sibling relationship as a source of support for survivors of sexual violence. Finally, as policies continue to develop around support of survivors of sexual violence, inclusion of siblings in family policies, outreach centers, and counseling services may be beneficial.
Asunto(s)
Delitos Sexuales , Hermanos , Revelación , Femenino , Humanos , Delitos Sexuales/psicología , Sobrevivientes/psicologíaRESUMEN
BACKGROUND/AIM: SARS-CoV-2 with a dramatical worldwide spread, impacted greatly daily life and healthcare. In order to avoid delay in cancer treatment, many strategies and measures were implemented. The Awake breast surgery was a strategy implemented in our Unit during the pandemic, aimed to reduce operatory room occupancy and increase the number of procedures performed during the daily surgical session. The aim of the study was to evaluate how the use of this strategy has changed before and after the advent of the COVID-19 pandemic, and its relative benefits. PATIENTS AND METHODS: This was a retrospective study analysing all patients subjected to breast conservative surgery for oncological disease from July 2018 to December 2021. RESULTS: Out of 498 patients enrolled in the study, 253 (50.8%) cases were treated before the pandemic and were designated as "pre-COVID-19" group. The remaining 245 (49.1%) cases were considered the "COVID-19" group. Cases of awake surgery in COVID-19 group were 141 (54.7%) vs. 84 (33.2%), p<0.001. Length of hospitalization and surgical time were comparable between the groups: relative p=0.188 and 0.264, respectively. Differently, operation room occupation was significantly shorter in the COVID-19 group, p<0.001; and number of outpatient surgical procedures was higher, p=0.0304. Multivariate analysis identified the period of surgery (OR=1.47) as a statistically significant factor, p=0.011, predictive of prolonged operatory room occupancy. CONCLUSION: Awake surgery was one of the strategies which made more operating rooms available and allowed avoiding further delays.
Asunto(s)
Neoplasias Encefálicas , COVID-19 , COVID-19/epidemiología , Humanos , Pandemias , Estudios Retrospectivos , SARS-CoV-2 , Listas de Espera , VigiliaRESUMEN
BACKGROUND/AIM: Corona virus infection dramatically spread worldwide during 2020 and extraordinary restrictions have been implemented in order to reduce viral transmission. These measures compelled a complete restructuring of the health system, including temporary cancer screening suspension and a significant slow-down in cancer diagnoses and treatments. CASE REPORT: We report five cases of extremely advanced breast cancer referred to our Department amid the COVID-19 pandemic. These patients exhibited a poor prognosis or worse quality of life due to their oncological disease. CONCLUSION: In our opinion, both the slow-down of diagnosis and treatment of oncological disease and anxiety over COVID-19 influenced this presentation. Moreover, other patients were unable to receive palliative care. Hopefully, these cases will not develop into extremely advanced-stage disease, and we will be able to provide at least the necessary palliative care.
Asunto(s)
Neoplasias de la Mama , COVID-19 , Ansiedad/diagnóstico , Ansiedad/epidemiología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Femenino , Humanos , Pandemias , Calidad de Vida , SARS-CoV-2RESUMEN
Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and approximately 1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.
Asunto(s)
Actinomycetales/enzimología , Celulosa/metabolismo , Celulosomas/metabolismo , Xilanos/metabolismo , Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Tallos de la Planta/metabolismo , Triticum/metabolismoRESUMEN
COVID-19 has been officially declared as a pandemic by the WHO. Italy was the first European country to be strongly affected by this outbreak. All elective and health promotion activities were reduced. Accordingly, Italian Breast Units and breast cancer (BC) screening programs scaled down significantly their activities. The aim of this study was to evaluate measures that could potentially reduce the clinical impact of COVID-19 on BC patients. Temporary recommendations are needed that could assist specialists in preventing COVID-19 infection and optimizing resources for diagnosis and treatment of BC patients.
Asunto(s)
Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/terapia , Infecciones por Coronavirus , Procedimientos Quirúrgicos Electivos/psicología , Hospitales Universitarios , Hospitales Urbanos , Mastectomía/psicología , Pandemias , Neumonía Viral , Negativa del Paciente al Tratamiento/psicología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/psicología , Neoplasias de la Mama/cirugía , COVID-19 , Carcinoma/diagnóstico por imagen , Carcinoma/psicología , Carcinoma/cirugía , Carcinoma/terapia , Carcinoma Intraductal no Infiltrante/diagnóstico por imagen , Carcinoma Intraductal no Infiltrante/psicología , Carcinoma Intraductal no Infiltrante/cirugía , Terapia Combinada , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/psicología , Diagnóstico Tardío , Manejo de la Enfermedad , Detección Precoz del Cáncer , Estrógenos , Femenino , Humanos , Mamografía , Tamizaje Masivo , Terapia Neoadyuvante , Neoplasias Hormono-Dependientes/diagnóstico por imagen , Neoplasias Hormono-Dependientes/psicología , Neoplasias Hormono-Dependientes/cirugía , Neoplasias Hormono-Dependientes/terapia , Pandemias/prevención & control , Neumonía Viral/epidemiología , Neumonía Viral/prevención & control , Neumonía Viral/psicología , Ciudad de Roma , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/psicología , Neoplasias de la Mama Triple Negativas/cirugía , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
BACKGROUND/AIM: Coronavirus disease is spreading worldwide. Due to fast transmission and high fatality rate drastic emergency restrictions were issued. During the lockdown, only urgent medical services are guaranteed. All non-urgent services, as breast cancer (BC) screening, are temporarily suspended. The potential of breast cancer screening programs in increasing the survival rate and decreasing the mortality rate has been widely confirmed. Suspension could lead to worse outcomes for breast cancer patients. Our study aimed to analyse the data and provide estimates regarding the temporary BC screening suspension. PATIENTS AND METHODS: Data regarding breast cancer and respective screening programs were achieved through literature research and analysis. RESULTS: Considering three different scenarios with respect to the lockdown's impact on breast cancer screening, we estimate that approximately 10,000 patients could have a missed diagnosis during these 3 months. Considering a 6-month period, as suggested by the Imperial college model, the number of patients who will not receive a diagnosis will rise to 16,000. CONCLUSION: Breast cancer screening should be resumed as soon as possible in order to avoid further breast cancer missed diagnosis and reduce the impact of delayed diagnosis.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Infecciones por Coronavirus/epidemiología , Detección Precoz del Cáncer , Tamizaje Masivo/tendencias , Neumonía Viral/epidemiología , Anciano , Betacoronavirus/patogenicidad , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/epidemiología , COVID-19 , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/virología , Femenino , Humanos , Mamografía , Persona de Mediana Edad , Pandemias , Neumonía Viral/complicaciones , Neumonía Viral/virología , SARS-CoV-2RESUMEN
BACKGROUND/AIM: Coronavirus-19 (COVID-19) pandemic outbreak is currently having a huge impact on medical resource allocation. Breast Cancer (BC) patients are concerned both with BC treatment and COVID-19. This study aimed to estimate the impact of anxiety among patients, caused by the spreading of COVID-19. PATIENTS AND METHODS: Between the 16th of January and the 20th of March 2020, we retrospectively enrolled 160 patients. Eighty-two patients with a suspected breast lesion (SBL) were divided into two groups: PRE-COVID-19-SBL and POST-COVID-19-SBL. Seventy-eight BC patients were divided into PRE-COVID-19-BC and POST-COVID-19-BC. Patient characteristics including age, marital status, SBL/BC diameter, personal and family history of BC, clinical stage and molecular subtype were recorded. Procedure Refusal (PR) and Surgical Refusal (SR) were also recorded with their reason. RESULTS: BC and SBL analysis showed no difference in pre-treatment characteristics (p>0.05). Both POST-COVID-19-SBL and POST-COVID-19-BC groups showed higher rates of PR and SR (p=0.0208, p=0.0065 respectively). Infection risk represented primary reason for refusal among POST-COVID-19 patients. CONCLUSION: COVID-19-related anxiety could affect patients' decision-making process.
Asunto(s)
Ansiedad/psicología , Biopsia con Aguja Gruesa/psicología , Neoplasias de la Mama/psicología , Infecciones por Coronavirus/psicología , Toma de Decisiones , Procedimientos Quirúrgicos Electivos/psicología , Miedo/psicología , Mastectomía/psicología , Neumonía Viral/psicología , Negativa del Paciente al Tratamiento/psicología , Anciano , Anciano de 80 o más Años , Ansiedad/etiología , Actitud Frente a la Salud , Biopsia con Aguja Gruesa/métodos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , COVID-19 , Femenino , Humanos , Italia , Mamografía , Persona de Mediana Edad , Pandemias , Estudios Retrospectivos , Factores Socioeconómicos , Ultrasonografía Mamaria , VacioRESUMEN
BACKGROUND/AIM: In 2016, in the United States, more than 50% of breast cancer (BC) cases were diagnosed in patients older than 60 years of age. Our study aimed to estimate the risk of locoregional recurrence (LR) in patients who underwent breast-conservative treatment (BCT), according to age. PATIENTS AND METHODS: This retrospective monocentric study analyzed 613 cases of patients who underwent BCT between 2003 and 2014. Patients were divided into groups according to age: Under70 (under 70 years old) and Over70 (above 70 years old). Margins width, histology results, prognostic and predictive factors were compared. Subgroup analysis was performed for patients who experienced LR. RESULTS: LR Incidence among Under70 and Over70 was 5.4% and 1.7%, respectively (p<0.01). Group Over70 is characterized by larger tumors and a lower Ki67 index (p<0.01). CONCLUSION: Operation time reduction, better aesthetic results and reduced LR risk support BCT. The Over70 group exhibited better outcomes in terms of LR despite larger tumor dimensions.
Asunto(s)
Neoplasias de la Mama/epidemiología , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Estudios de Casos y Controles , Femenino , Humanos , Mastectomía Segmentaria , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Cuidados Posoperatorios , Pronóstico , Receptores de Estrógenos/metabolismo , Estudios Retrospectivos , RiesgoRESUMEN
BACKGROUND/AIM: Extraordinary restrictions aimed to limit Sars-CoV-2 spreading; they imposed a total reorganization of the health-system. Oncological treatments experienced a significant slowdown. The aim of our multicentric retrospective study was to evaluate screening suspension and surgical treatment delay during COVID-19 and the impact on breast cancer presentation. PATIENTS AND METHODS: All patients who underwent breast surgery from March 11, 2020 to May 30, 2020 were evaluated and considered as the Lockdown group. These patients were compared with similar patients of the previous year, the Pre-Lockdown group. RESULTS: A total of 432 patients were evaluated; n=223 and n=209 in the Lockdown and Pre-lockdown-groups, respectively. At univariate analysis, waiting times, lymph-nodes involvement and cancer grading, showed a statistically significant difference (p<0.05). Multivariate analysis identified waiting-time on list (OR=1.07) as a statistically significant predictive factor of lymph node involvement. CONCLUSION: Although we did not observe a clinically evident difference in breast cancer presentation, we reported an increase in lymph node involvement.
Asunto(s)
Neoplasias de la Mama/epidemiología , COVID-19/epidemiología , Pandemias , SARS-CoV-2/patogenicidad , Adulto , Anciano , Anciano de 80 o más Años , Axila/patología , Axila/cirugía , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/virología , COVID-19/complicaciones , COVID-19/virología , Femenino , Humanos , Escisión del Ganglio Linfático , Ganglios Linfáticos , Metástasis Linfática , Mastectomía , Persona de Mediana Edad , Estudios Retrospectivos , Biopsia del Ganglio Linfático CentinelaRESUMEN
BACKGROUND/AIM: Despite the large amount of clinical data available of Coronavirus-19 (COVID-19), not many studies have been conducted about the psychological toll on Health Care Workers (HCWs). PATIENTS AND METHODS: In this multicentric descriptive study, surveys were distributed among 4 different Breast Cancer Centers (BCC). BCCs were distinguished according to COVID-19 tertiary care hospital (COVID/No-COVID) and district prevalence (DP) (High vs. Low). DASS-21 score, PSS score and demographic data (age, sex, work) were evaluated. RESULTS: A total of 51 HCWs were analyzed in the study. Age, work and sex did not demonstrate statistically significant values. Statistically significant distribution was found between DASS-21-stress score and COVID/No-COVID (p=0.043). No difference was found in the remaining DASS-21 and PSS scores, dividing the HCWs according to COVID-19-hospital and DP. CONCLUSION: Working in a COVID-19-hospital represents a factor that negatively affects psychosocial well-being. However, DP seems not to affect the psychosocial well-being of BCC HCWs. During the outbreak, psychological support for low risk HCWs should be provided regardless DP.
Asunto(s)
Neoplasias de la Mama , Instituciones Oncológicas , Infecciones por Coronavirus/psicología , Enfermedades Profesionales/prevención & control , Grupo de Atención al Paciente , Personal de Hospital/psicología , Neumonía Viral/psicología , Adulto , COVID-19 , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/transmisión , Depresión/epidemiología , Depresión/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Laboral/epidemiología , Estrés Laboral/etiología , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/transmisión , Prevalencia , Equipos de Seguridad/provisión & distribución , Sistemas de Apoyo Psicosocial , Ciudad de Roma , Índice de Severidad de la Enfermedad , Centros de Atención Terciaria , Incertidumbre , Carga de TrabajoRESUMEN
We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant "cellulosomized" enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
Asunto(s)
Actinomycetales/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Celulasa/genética , Celulasa/metabolismo , Celulosomas/genética , Celulosomas/metabolismo , Celulosa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Cellulosomes are multi-enzyme complexes produced by certain anaerobic bacteria that exhibit efficient degradation of plant cell wall polysaccharides. To understand their enhanced levels of hydrolysis, we are investigating the effects of converting a free-cellulase system into a cellulosomal one. To achieve this end, we are replacing the cellulose-binding module of the native cellulases, produced by the aerobic bacterium Thermobifida fusca, with a cellulosome-derived dockerin module of established specificity, to allow their incorporation into defined "designer cellulosomes". In this communication, we have attached divergent dockerins to the two exoglucanases produced by T. fusca exoglucanase, Cel6B and Cel48A. The resultant fusion proteins were shown to bind efficiently and specifically to their matching cohesins, and their activities on several different cellulose substrates were compared. The lack of a cellulose-binding module in Cel6B had a deleterious effect on its activity on crystalline substrates. In contrast, the dockerin-bearing family-48 exoglucanase showed increased levels of hydrolytic activity on carboxymethyl cellulose and on both crystalline substrates tested, compared to the wild-type enzyme. The marked difference in the response of the two exoglucanases to incorporation into a cellulosome, suggests that the family-48 cellulase is more appropriate than the family-6 enzyme as a designer cellulosome component.
Asunto(s)
Actinomycetales/enzimología , Celulasas/metabolismo , Celulosa/metabolismo , Celulosomas/enzimología , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Hidrólisis , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Especificidad por Sustrato , CohesinasRESUMEN
Self assembly is a prerequisite for fabricating nanoscale structures. Here we present a new fusion protein based on the stress-responsive homo-oligomeric protein, SP1. This ring-shaped protein is a highly stable homododecamer, which can be potentially utilized to self-assemble different modules and enzymes in a predicted and oriented manner. For that purpose, a cohesin module (a component of the bacterial cellulosome) was selected, its gene fused in-frame to SP1, and the fusion protein was expressed in Escherichia coli. The cohesin module, specialized to incorporate different enzymes through specific recognition of a dockerin modular counterpart, is used to display new moieties on the SP1 scaffold. The SP1 scaffold displayed 12 active cohesin modules and specific binding to a dockerin-fused cellulase enzyme from Thermobifida fusca. Moreover, we found a significant increase in specific activity of the scaffold-displayed enzymes.
Asunto(s)
Celulasa/metabolismo , Nanotecnología , Proteínas Recombinantes de Fusión/metabolismo , Catálisis , Proteínas de Ciclo Celular/metabolismo , Cromatografía en Gel , Proteínas Cromosómicas no Histona/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Nucleares/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , CohesinasRESUMEN
Protein molecular scaffolds are attracting interest as natural candidates for the presentation of enzymes and acceleration of catalytic reactions. We have previously reported evidence that the stable protein 1 (SP1) from Populustremula can be employed as a molecular scaffold for the presentation of either catalytic or structural binding (cellulosomal cohesin) modules. In the present work, we have displayed a potent exoglucanase (Cel6B) from the aerobic cellulolytic bacterium, Thermobifida fusca, on a cohesin-bearing SP1 scaffold. For this purpose, a chimaeric form of the enzyme, fused to a cellulosomal dockerin module, was prepared. Full incorporation of 12 dockerin-bearing exoglucanase molecules onto the cohesin-bearing scaffold was achieved. Cellulase activity was tested on two cellulosic substrates with different levels of crystallinity, and the activity of the scaffold-linked exoglucanase was significantly reduced, compared to the free dockerin-containing enzyme. However, addition of relatively low concentrations of a free wild-type endoglucanase (T. fusca Cel5A) that bears a cellulose-binding module, in combination with the complexed exoglucanase resulted in a marked rise in activity on both cellulosic substrates. The endoglucanase cleaves internal sites of the cellulose chains, and the new chain ends of the substrate were now readily accessible to the scaffold-borne exoglucanase, thereby resulting in highly effective, synergistic degradation of cellulosic substrates.
Asunto(s)
Biotecnología/métodos , Celulasa/metabolismo , Celulosa/metabolismo , Nanoestructuras/química , Proteínas de Ciclo Celular/metabolismo , Cromatografía en Gel , Proteínas Cromosómicas no Histona/metabolismo , Clostridium/metabolismo , Nanoestructuras/ultraestructura , Proteínas de Plantas/ultraestructura , Populus/metabolismo , Proteínas Recombinantes/metabolismo , CohesinasRESUMEN
Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein-protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes' CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.
RESUMEN
Designer cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fusca glycoside hydrolases. Two T. fusca cellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fusca xylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fusca enzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome.
Asunto(s)
Actinomycetales/enzimología , Celulasa/genética , Celulasa/metabolismo , Celulosomas/genética , Celulosomas/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Actinomycetales/genética , Biotecnología/métodos , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triticum/metabolismoRESUMEN
Inteins are autocatalytic protein domains that post-translationally excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate genes at their intein domain. Such naturally occurring split inteins rapidly self-associate and reconstitute protein-splicing activity in trans. Here, we analyze the in vitro protein-splicing activity of three naturally split inteins from diverse cyanobacteria: Oscillatoria limnetica, Thermosynechococcus vulcanus, and Nostoc sp. PCC7120. N- and C-terminal halves of these split inteins were mixed in nine combinations, resulting in three endogenous (wild-type) and six exogenous combinations. Protein splicing was detected in all split-intein combinations, despite a 30-50% sequence variation between the homologous proteins. Splicing activity proceeded under a variety of conditions, including the presence of denaturants and reductants and high temperature, ionic strength, and viscosity. Still, in a high concentration of salt (2 M) or urea (6 M), specific combinations spliced significantly better than others. Additionally, copper ions were found to inhibit trans splicing in a reversible double-lock reaction. Our comparative analysis of naturally split inteins in endogenous and exogenous combinations demonstrates the modularity of trans protein-splicing elements and their robust activity. It suggests tight interactions between split-intein halves and conditions for modifying the specificity of intein parts. These results promote the biotechnological use of split inteins for controlled assembly of protein fragments either in vivo or in vitro and under moderate or extreme conditions.
Asunto(s)
Cianobacterias/metabolismo , Inteínas , Empalme de Proteína/fisiología , Secuencia de Aminoácidos , Cianobacterias/clasificación , Datos de Secuencia Molecular , Desnaturalización Proteica , Temperatura , Trans-EmpalmeRESUMEN
Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analysing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnology.