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BACKGROUND: Plasma exchange (PLEX) therapy is indicated for several disorders. The 5% albumin is often used as a sole replacement fluid during most PLEX. However, each 1.0 plasma volume exchange depletes coagulation factors by ~65%. Although most coagulation factors recover to hemostatic levels within 24 h post-PLEX, fibrinogen requires 48-72 h to recover. Fibrinogen is the key coagulation protein for hemostasis. Therefore, fibrinogen is often monitored during the acute course of PLEX, and plasma is supplemented to prevent bleeding if fibrinogen is <100 mg/dL. STUDY DESIGN AND METHODS: We conducted a prospective, single-center, observational study to evaluate bleeding risk in adults who received an acute course of PLEX with a fibrinogen level of 80-100 mg/dL without plasma supplementation during the procedure or before central venous catheter removal. The study group was compared to patients with plasma fibrinogen >100 mg/dL. RESULTS: Among the 275 patients who received 1406 PLEXes, 62 patients (23%) who underwent 323 PLEXes met the inclusion criteria, and only 2 (3%) patients had bleeding while on oral anticoagulants. In contrast, out of 275 patients, 143 (52%) with fibrinogen levels >100 mg/dL received 751 PLEX treatments, and bleeding occurred in 2 (1%) while on low-molecular-weight heparin. CONCLUSIONS: Our findings suggest that a pre-procedure fibrinogen threshold of 80-100 mg/dL without plasma supplementation does not increase bleeding risk unless patients were on anticoagulation.
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Fibrinógeno , Hemorragia , Intercambio Plasmático , Humanos , Intercambio Plasmático/efectos adversos , Intercambio Plasmático/métodos , Fibrinógeno/análisis , Fibrinógeno/metabolismo , Estudios Prospectivos , Masculino , Femenino , Persona de Mediana Edad , Hemorragia/etiología , Hemorragia/sangre , Hemorragia/terapia , Anciano , Adulto , Factores de RiesgoRESUMEN
BACKGROUND: Some patients with end stage renal disease are or will become narcotic-dependent. Chronic narcotic use is associated with increased graft loss and mortality following kidney transplantation. We aimed to compare the efficacy of continuous flow local anesthetic wound infusion pumps (CFLAP) with patient controlled analgesia pumps (PCA) in reducing inpatient narcotic consumption in patients undergoing kidney transplantation. MATERIALS AND METHODS: In this single-center, retrospective analysis of patients undergoing kidney transplantation, we collected demographic and operative data, peri-operative outcomes, complications, and inpatient oral morphine milligram equivalent (OME) consumption. RESULTS: Four hundred and ninety-eight patients underwent kidney transplantation from 2020 to 2022. 296 (59%) historical control patients received a PCA for postoperative pain control and the next 202 (41%) patients received a CFLAP. Median age [53.5 vs. 56.0 years, p = .08] and BMI [29.5 vs. 28.9 kg/m2, p = .17] were similar. Total OME requirement was lower in the CFLAP group [2.5 vs. 34 mg, p < .001]. Wound-related complications were higher in the CFLAP group [5.9% vs. 2.7%, p = .03]. Two (.9%) patients in the CFLAP group experienced cardiac arrhythmia due to local anesthetic toxicity and required lipid infusion. CONCLUSIONS: Compared to PCA, CFLAP provided a 93% reduction in OME consumption with a small increase in the wound-related complication rate. The utility of local anesthetic pumps may also be applicable to patients undergoing any unilateral abdominal or pelvic incision.
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Analgesia , Trasplante de Riñón , Humanos , Anestésicos Locales , Estudios Retrospectivos , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/etiología , Trasplante de Riñón/efectos adversos , Analgésicos Opioides/uso terapéutico , Narcóticos , Analgesia/efectos adversosRESUMEN
INTRODUCTION: The study purpose was to review retrospectively our single-center experience transplanting kidneys from deceased donors (DD) with acute kidney injury (AKI) according to terminal serum creatinine (tSCr) level. METHODS: AKI kidneys were defined by a doubling of the DD's admission SCr and a tSCr ≥ 2.0 mg/dL. RESULTS: From 1/07 to 11/21, we transplanted 236 AKI DD kidneys, including 100 with a tSCr ≥ 3.0 mg/dL (high SCr AKI group, mean tSCr 4.2 mg/dL), and the remaining 136 from DDs with a tSCr of 2.0-2.99 mg/dL (lower SCr AKI group, mean tSCr 2.4 mg/dL). These two AKI groups were compared to 996 concurrent control patients receiving DD kidneys with a tSCr < 1.0 mg/dL. Mean follow-up was 69 months. Delayed graft function (DGF) rates were 51% versus 46% versus 29% (p < 0.0001), and 5-year patient and death-censored kidney graft survival rates were 96.8% versus 83.5% versus 82.2% (p = 0.002) and 86.7% versus 77.8% versus 78.8% (p = 0.18) in the high tSCr AKI versus lower tSCr AKI versus control groups, respectively. CONCLUSIONS: Despite a higher incidence of DGF, patients receiving kidneys from DDs with tSCr levels ≥3.0 mg/dL have acceptable medium-term outcomes compared to either AKI DDs with a lower tSCr or DDs with a tSCr < 1.0 mg/dL.
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Lesión Renal Aguda , Supervivencia de Injerto , Trasplante de Riñón , Donantes de Tejidos , Humanos , Lesión Renal Aguda/etiología , Trasplante de Riñón/efectos adversos , Masculino , Femenino , Estudios Retrospectivos , Persona de Mediana Edad , Donantes de Tejidos/provisión & distribución , Estudios de Seguimiento , Pronóstico , Tasa de Supervivencia , Rechazo de Injerto/etiología , Tasa de Filtración Glomerular , Factores de Riesgo , Funcionamiento Retardado del Injerto/etiología , Adulto , Pruebas de Función Renal , Complicaciones Posoperatorias/etiología , Creatinina/sangre , Índice de Severidad de la Enfermedad , Fallo Renal Crónico/cirugíaRESUMEN
INTRODUCTION: The study aim was to analyze the presentation, management, and follow-up of renal transplant patients developing bladder calculi. METHODS: Patients who underwent renal transplant with postoperative follow-up at our institution were retrospectively analyzed (1984-2023) to assess for the development of posttransplant bladder stones. All bladder stones were identified by computerized tomography imaging and stone size was measured using this imaging modality. RESULTS: The prevalence of bladder calculi post-renal transplantation during the study window was 0.22% (N = 20/8,835) with a median time to bladder stone diagnosis of 13 years posttransplant. Of all bladder stone patients, 6 (30%) received deceased donor and 14 (70%) living donor transplants. There were 11 patients with known bladder stone composition available; the most common being calcium oxalate (N = 6). Eleven (55%) patients had clinical signs or symptoms (most commonly microhematuria). Fourteen of the bladder stone cohort patients (70%) underwent treatment including cystolitholapaxy in 12 subjects. Of these 14 patients, 9 (64%) were found to have nonabsorbable suture used for their ureteroneocystostomy closure. CONCLUSIONS: The prevalence of bladder stones post-renal transplant is low. The utilization of nonabsorbable suture for ureteral implantation was the main risk factor identified in our series. This technique is no longer used at our institution. Other factors contributing to bladder stone formation in this population warrant identification.
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INTRODUCTION: Long-term outcomes of kidney transplantation from deceased donors (DDKTs) with terminal acute kidney injury (AKI) are not well defined. METHODS: Single center retrospective review of DDKTs from 1/31/07-12/31/19. AKI kidneys were defined by a doubling of the donor's admission serum creatinine (SCr) level AND a terminal SCr ≥2.0 mg/dl. RESULTS: A total of 188 AKI DDKTs were performed, including 154 from brain-dead standard criteria donors (SCD). Mean donor age was 36 years and mean Kidney Donor Profile Index was 50%; mean admission and terminal SCr levels were 1.3 and 3.1 mg/dl, respectively. With a mean follow-up of 94 months (median 89 months), overall patient (both 71.3%) and graft survival (54% AKI vs. 57% non-AKI) rates were comparable to concurrent DDKTs from brain-dead non-AKI SCDs (n = 769). Delayed graft function (DGF) was higher in AKI kidney recipients (47% vs. 20% non-AKI DDKTs, p < .0001). DGF was associated with lower graft survival in recipients of both AKI and non-AKI SCD kidneys but the impact was earlier and more pronounced in non-AKI recipients. CONCLUSIONS: Despite having more than twice the incidence of DGF, kidneys from deceased donors with terminal AKI have long-term outcomes comparable to non-AKI SCD kidneys and represent a safe and effective method to expand the donor pool.
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Lesión Renal Aguda , Trasplante de Riñón , Humanos , Adulto , Trasplante de Riñón/efectos adversos , Donantes de Tejidos , Riñón , Supervivencia de Injerto , Estudios Retrospectivos , Muerte Encefálica , Funcionamiento Retardado del Injerto/etiologíaRESUMEN
INTRODUCTION: There is limited experience transplanting kidneys from either expanded criteria donors (ECD) or donation after circulatory death (DCD) deceased donors with terminal acute kidney injury (AKI). METHODS: AKI kidneys were defined by a donor terminal serum creatinine level >2.0 mg/dL whereas non-ideal deceased donor (NIDD) kidneys were defined as AKI/DCD or AKI/ECDs. RESULTS: From February 2007 to March 2023, we transplanted 266 single AKI donor kidneys including 29 from ECDs, 29 from DCDs (n = 58 NIDDs), and 208 from brain-dead standard criteria donors (SCDs). Mean donor age (43.7 NIDD vs. 33.5 years SCD), KDPI (66% NIDD vs. 45% SCD), and recipient age (57 NIDD vs. 51 years SCD) were higher in the NIDD group (all p < .01). Mean waiting times (17.8 NIDD vs. 24.2 months SCD) and dialysis duration (34 NIDD vs. 47 months SCD) were shorter in the NIDD group (p < .05). Delayed graft function (DGF, 48%) and 1-year graft survival (92.7% NIDD vs. 95.9% SCD) was similar in both groups. Five-year patient and kidney graft survival rates were 82.1% versus 89.9% and 82.1% versus 75.2% (both p = NS) in the NIDD versus SCD groups, respectively. CONCLUSIONS: The use of kidneys from AKI donors can be safely liberalized to include selected ECD and DCD donors.
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Lesión Renal Aguda , Trasplante de Riñón , Humanos , Estudios Retrospectivos , Cadáver , Donantes de Tejidos , Riñón , Lesión Renal Aguda/etiología , Supervivencia de Injerto , Recompensa , Resultado del TratamientoRESUMEN
AIM: The influence of dialysis modality and duration on outcomes following simultaneous pancreas-kidney transplantation (SPKT) remains uncertain. METHODS: We performed a single-center retrospective review in 255 SPKT recipients according to dialysis modality (55 preemptive/no dialysis-ND, 70 peritoneal dialysis-PD, 130 hemodialysis-HD) and duration (55 none, 137 < 2 years, 41 2-4 years, 22 > 4 years). RESULTS: Mean follow-up was 9.4 years (median 9.2 years). Early (3-month) relaparotomy rate (20% ND vs. 36% PD/HD, p = .03) was lower in ND patients. There were no differences in early graft loss, patient survival, overall or death-censored kidney or pancreas graft survival rates (GSR) at 1 or 10 years follow-up. When analyzing dialysis duration, there were no differences in rates of pancreas thrombosis or early pancreas graft loss. Kidney delayed graft function (DGF) was lower in the ND/short dialysis groups combined (1.0%), compared to the intermediate/long dialysis groups combined (9.5%, p = .003). Early relaparotomy rates were higher with longer duration of dialysis (p = .045 between ND and >4 years of dialysis). Patient survival in the long dialysis group was 50% compared to 69.5% in the other three groups combined (p = .09). However, both overall and death-censored kidney and pancreas GSR were comparable. CONCLUSIONS: Preemptively transplanted patients had a lower incidence of kidney DGF and relaparotomy whereas patient survival was slightly lower with longer dialysis vintage prior to SPKT. Dialysis modality and duration did not influence either overall or death-censored pancreas or kidney GSR in patients with short waiting times, low KDPI donor organs, and dialysis duration up to 4 years.
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Trasplante de Riñón , Trasplante de Páncreas , Diálisis Peritoneal , Humanos , Resultado del Tratamiento , Diálisis Renal , Estudios Retrospectivos , Páncreas , Supervivencia de InjertoRESUMEN
BACKGROUND: Hyperhemolysis syndrome (HHS) is an uncommon transfusion reaction described in several hematologic disorders, including sickle cell disease (SCD). HHS is characterized by a decline in hemoglobin (Hb) values below pre-transfusion levels following transfusion of red blood cells (RBCs), coupled with laboratory markers consistent with hemolysis. The proposed pathophysiologic mechanisms underlying HHS include increased phosphatidylserine expression, macrophage activation, and complement dysregulation. Many pathophysiologic mechanisms thought to contribute to HHS have been similarly described in cases of severe COVID-19. CASE REPORT: A 28-year-old male with a history of HbSS presented with shortness of breath, right-sided chest pain, and a two-day history of fever. Polymerase chain reaction (PCR) detected SARS-CoV-2 infection with the omicron variant. The patient required an RBC transfusion (pre-transfusion hemoglobin [Hb]5.8 g/dL) with an immediate post-transfusion Hb of 6.3 g/dL. However, Hb rapidly declined to 1.7 g/dL, and lactate dehydrogenase (LDH) rose to 8701 u/L. The absolute reticulocyte count of 538 × 109/L correspondingly fell to 29 × 109/L. Despite additional RBC transfusions and initiation of immunosuppressive therapy, he expired on Day 9(D9). CONCLUSION: Given the similarities in their proposed pathophysiology, patients with SCD and concomitant SARS-CoV-2 infection may be predisposed to developing HHS.
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Anemia de Células Falciformes , COVID-19 , Masculino , Humanos , Adulto , COVID-19/complicaciones , SARS-CoV-2 , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/terapia , Hemólisis , Síndrome , HemoglobinasRESUMEN
BACKGROUND: Acquired Hemophilia A (AHA) is a rare autoimmune disorder associated with the development of autoantibodies against factor VIII (FVIII). Although obtaining hemostatic control through the use of recombinant factor VIIa, activated prothrombin complex concentrate and recombinant porcine FVIII are cornerstones in the clinical management of AHA, these therapies have several disadvantages, including a higher risk for the development of thromboembolic events, unpredictable efficacy and short half-lives. While emicizumab has been FDA licensed for use in bleeding prophylaxis for patients with Congenital Hemophilia A (CHA) with and without inhibitors, it has not been approved for use in AHA, with only a few reports describing its use in this context. CASE REPORT: We report our experience with the use of emicizumab in an 83-year old male with AHA, complicated by the onset of atrial fibrillation following admission, drug-induced thrombocytopenia, infectious complications, and the identification of a low-grade lymphoproliferative disorder, in which emicizumab prophylaxis was used for bleeding prophylaxis in the context of persistently elevated inhibitor titers without evidence of thrombotic events or thrombotic microangiopathy.
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Anticuerpos Biespecíficos , Hemofilia A , Hemostáticos , Masculino , Porcinos , Animales , Hemofilia A/tratamiento farmacológico , Factor VIII/uso terapéutico , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Hemorragia/tratamiento farmacológico , Hemorragia/prevención & control , Hemostáticos/uso terapéuticoRESUMEN
BACKGROUND: Appendiceal cancers represent a diverse group of malignancies with varying biological behavior. The significance of lymph node metastases in relation to long-term survival and chemotherapy response is poorly defined. METHODS: The National Cancer Database was queried to find patients diagnosed with appendiceal cancer from 1998 to 2012. Kaplan-Meier curves and multivariable Cox regression analyses were used to study the association between lymph node status and overall survival. Stage IV patients were excluded. RESULTS: The rate of nodal positivity of the 9841 patients with known node status was: signet ring 47.4%, carcinoid 42.3%, nonmucinous adenocarcinoma 28.8%, goblet cell 21.9%, and mucinous adenocarcinoma 20.4%. Node-positive patients had worse long-term survival for all subtypes with the exception of carcinoid tumors (p < 0.001). The strongest association was for signet cell and goblet cell. Adjuvant chemotherapy in node-positive patients improved survival for mucinous, nonmucinous, and signet ring cell histology (p < 0.01), but not for goblet cell. CONCLUSIONS: Nodal involvement in patients with appendiceal cancer varies in incidence, association with adverse survival, and response to systemic therapy. Individualized treatment algorithms for the management of the subtypes of appendiceal cancer are needed.
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Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Neoplasias del Apéndice/mortalidad , Neoplasias del Apéndice/patología , Adenocarcinoma/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apendicectomía , Neoplasias del Apéndice/tratamiento farmacológico , Quimioterapia Adyuvante , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Tasa de Supervivencia , Adulto JovenRESUMEN
OBJECTIVES: Hypertriglyceridemia-induced acute pancreatitis (HTG-AP) accounts for 1 to 10% of pancreatitis cases, and is associated with a more severe clinical course. Therapeutic plasma exchange (TPE) is a potential treatment option for quickly lowering plasma triglycerides (TG). Current ASFA guidelines define HTG-AP as a Category III disorder, indicating the role of apheresis is not firmly established. Here, we examine clinical data regarding its effectiveness on morbidity and mortality in patients with HTG-AP presenting with severely elevated plasma triglycerides (>4000 mg/dl). METHODS: We retrospectively examined clinical data and outcomes from 67 consecutive episodes of HTG-AP over a 5-year period in which either medical management alone or medical management plus adjunct TPE was employed to reduce plasma triglycerides. RESULTS: 16/67 admissions involved TPE, initiated at a mean of 0.7 days from the time of presentation, while 51 received medical management alone. After only one TPE procedure, the mean TG values decreased from 4103 to 1045 mg/dl (a reduction of 74.7%), and those receiving TPE reached plasma TG < 1000 mg/dl 0.99 days faster than the medical group. One patient in the TPE group died. However, when excluding patients with hospital courses complicated by multiple organ dysfunction, there was no significant difference in mortality or hospital length of stay (LOS) between the groups. CONCLUSIONS: In uncomplicated cases of HTG-AP with an absence of multiorgan dysfunction, there is no significant benefit to either mortality or LOS when adding adjunct TPE to medical management, even when patients present with severely elevated levels of TG.
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Hipertrigliceridemia/complicaciones , Pancreatitis/etiología , Pancreatitis/terapia , Intercambio Plasmático , Triglicéridos/sangre , Adulto , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Pancreatitis/sangre , Intercambio Plasmático/efectos adversos , Estudios RetrospectivosRESUMEN
BACKGROUND: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has been associated with increased postoperative complications and a prolonged length of stay (LOS). We report on our experience following implementation of an Enhanced Recovery After Surgery (ERAS) program for CRS and HIPEC. METHODS: Patients were divided into pre- and post-ERAS groups. Modifications in the ERAS group included routine use of transversus abdominis plane blocks, intra- and postoperative fluid restriction, and minimizing the use of narcotics, drains, and nasogastric tubes. RESULTS: Of a total of 130 procedures, 49 (38%) were in the pre-ERAS group and 81 (62%) were in the ERAS group. Mean LOS was reduced from 10.3 ± 8.9 days to 6.9 ± 5.0 days (p = 0.007) and the rate of grade III/IV complications was reduced from 24 to 15% (p = 0.243) following ERAS implementation. The ERAS group received less intravenous fluid during hospitalization (19.2 ± 18.7 L vs. 32.8 ± 32.5 L, p = 0.003) and used less opioids than the pre-ERAS group (median of 159.7 mg of oral morphine equivalents vs. 272.6 mg). There were no significant changes in the rates of 30-day readmission or acute kidney injury between the two groups (p = non-significant). On multivariable analyses, ERAS was significantly associated with a reduction in LOS (- 2.89 days, 95% CI - 4.84 to - 0.94) and complication rates (odds ratio 0.22, 95% CI 0.08-0.57). CONCLUSIONS: Implementation of an ERAS program for CRS and HIPEC is associated with a reduction in overall intravenous fluids, postoperative narcotic use, complication rates, and LOS.
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Procedimientos Quirúrgicos de Citorreducción , Recuperación Mejorada Después de la Cirugía , Hipertermia Inducida , Neoplasias/mortalidad , Neoplasias/terapia , Adulto , Anciano , Analgésicos Opioides/uso terapéutico , Antineoplásicos/uso terapéutico , Terapia Combinada , Femenino , Fluidoterapia/estadística & datos numéricos , Humanos , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Readmisión del Paciente , Complicaciones Posoperatorias/epidemiología , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415-507 of TER1. A ter1-Δ415-507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1-TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1-Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1Δ. We propose that the Est1-Ccq1 interaction is critical for telomerase recruitment, while the Est1-TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.
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Proteínas de Unión al ADN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Modelos Moleculares , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , ARN , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.
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ADN Helicasas/metabolismo , Replicación del ADN , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , ARN Polimerasa III/genética , ARN Polimerasa II/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Transcripción Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN de Transferencia/genética , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.
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Alelos , Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Proteína 9 Asociada a CRISPR/genética , Heterocigoto , Mutación , Fenotipo , Edición de ARNRESUMEN
The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.
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Elementos Aisladores/genética , ARN/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Telomerasa/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Proteínas Relacionadas con la Autofagia , Secuencia de Bases , Western Blotting , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Hibridación Fluorescente in Situ , Modelos Genéticos , Mutación , Unión Proteica , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Reversa , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Ácido Nucleico , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Moldes Genéticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Research suggests sleep disturbance plays a role in depression and risk for suicidal behavior (i.e., ideation, attempts, death by suicide). How sleep disturbance affects suicide risk is unclear and one's ability to perform activities of daily living (ADLs) may help explain this relation. This study examined associations between sleep problems, ADLs, and either depressive symptoms or suicide risk among older adults. We hypothesized that ADLs would mediate relations between sleep problems and depressive symptoms and suicide risk. METHOD: Participants (N = 134; age ≥65) were recruited through Amazon's Mechanical Turk. Participants completed questionnaires that assessed insomnia symptoms, nightmares, ADLs, depressive symptoms, and suicidal behaviors. RESULTS: Nightmares were associated with depressive symptoms and suicide risk but not independently associated with ADLs. Insomnia symptoms were associated with depressive symptoms, suicide risk, and ADLs. ADLs mediated the relation between insomnia symptoms and depressive symptoms. The insomnia symptom-suicidal behavior relation and the nightmare-suicidal behavior relation were significantly mediated by a pathway containing ADLs and depressive symptoms. DISCUSSION: ADLs help explain how insomnia symptoms and nightmares confer suicide risk among older adults, either independently or in association with depressive symptoms. CLINICAL IMPLICATIONS: Practitioners should attend to ADL performance when treating older adults with insomnia and depression.
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Actividades Cotidianas/psicología , Depresión/complicaciones , Trastornos del Inicio y del Mantenimiento del Sueño/complicaciones , Anciano , Estudios Transversales , Depresión/psicología , Sueños/psicología , Humanos , Factores de Riesgo , Autoinforme , Índice de Severidad de la Enfermedad , Sueño/fisiología , Trastornos del Inicio y del Mantenimiento del Sueño/psicología , Ideación Suicida , Encuestas y CuestionariosRESUMEN
BACKGROUND: Schizoaffective disorder (SAD) has routinely exhibited poor diagnostic accuracy and reliability. In addition to phenomenological problems with the definition of SAD, the way in which clinicians represent the symptoms of the disorder could contribute to its poor diagnostic outcomes. PURPOSE: The present study sought to examine clinicians' representations of SAD compared to schizophrenia (SCZ), bipolar disorder with psychotic features (BiPD-PSY), and major depressive disorder with psychotic features (MDD-PSY). METHOD: Participants (N=113) were clinicians recruited via email as part of a larger study. They were randomly assigned to either select symptoms from a predetermined criteria list or freely list features of the disorders based on their own mental representations. RESULTS: Participants' conceptualizations of SAD were not entirely congruent with DSM-5 criteria; they conceptualized it as less psychotic than SCZ and less affective than the two mood disorder tasks. SAD was conceptualized as significantly more depressive than manic. CONCLUSIONS: This study's findings support the notion that clinicians' conceptualizations of SAD are not entirely congruent with its DSM-5 criteria, which could contribute to diagnostic difficulties.