Citations

ABSTRACT Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii . Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii , which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti- Pneumocystis agents against human P. jirovecii . Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109 , Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109 -null yeast. Recombinant PjRtt109 is an active HAT in vitro , with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro . PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro . Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti- Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP.

AbstractBackgroundThere has been increasing interest in using protease inhibitors with pegylated interferon and ribavirin to treat recurrent hepatitis C (HCV) disease in liver transplant recipients.MethodsWe retrospectively evaluated the safety and efficacy in liver transplant recipients treated for recurrent hepatitis C genotype 1 with the combination of peginterferon, ribavirin and boceprevir.ResultsTwenty liver transplant recipients were treated for recurrent hepatitis C. Baseline alanine aminotransferase, total bilirubin and HCV RNA values (± SD) were 67.5 (±50.9) mg/dl, 1.78 (±1.99) U/L, and 16 955 510 (±21 620 675) IU/ml. Anaemia was a common adverse event requiring epoetin in 16 of 20 recipients and ribavirin dose reductions in 17 of 20 recipients. One‐third of recipients required a blood transfusion. Filgrastim was used in 11 of 20 patients (55%) and eltrombopag in two of 20 recipients (10%) over the course of treatment. Serum creatinine level increased significantly from a baseline value of 1.33 mg/dl to 1.59 mg/dl at week 20 of boceprevir (P < 0.005). The overall sustained viral response (SVR) was 50%. Of the 14 patients who had a viral load less than 1000 IU/ml at week 4 of boceprevir, the SVR was 71%. The SVR was 83% of the 11 patients who had undetectable viral levels at week 4 of boceprevir.ConclusionsAntiviral therapy utilizing boceprevir in liver transplant recipients requires close monitoring. Anaemia and neutropenia were common requiring growth factors in most recipients. On‐treatment viral responses appear promising but long‐term data are needed.

Abstract Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a “do-it-yourself” basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.

Objective To evaluate the relationship between partial nephrectomy ( PN ) and hospital availability of robot‐assisted surgery from a population‐based cohort in the USA . Methods After merging the Nationwide Inpatient Sample ( NIS ) and the A merican Hospital Association survey from 2006 to 2008, we identified 21 179 patients who underwent either PN or radical nephrectomy ( RN ) for renal cell carcinoma ( RCC ). The primary outcome assessed was the type of nephrectomy performed. Multivariable logistic regression identified the patient and hospital characteristics associated with receipt of PN . Results We identified 4832 (22.8%) and 16 347 (77.2%) patients who were treated for RCC with PN and RN , respectively. On multivariable analysis, patients were more likely to receive PN at academic centres (odds ratio [ OR ] 2.77; P < 0.001), urban centres ( OR 3.66; P < 0.001) and A merican College of Surgeons ( ACOS )‐designated cancer centres ( OR : 1.10; P < 0.05) compared with non‐academic, rural and non‐ ACOS ‐designated cancer centre hospitals, respectively. Robot‐assisted surgery availability at a hospital was also associated with a higher adjusted odds of PN compared with centres without that availability ( OR 1.28; P < 0.001). Conclusions Although academic and urban locations are establi s hed factors that affect the receipt of PN for RCC , the availability of robot‐assisted surgery at a hospital was also independently associated with higher use of PN . Our results are informative in identifying other key hospital characteristics which may facilitate greater adoption of PN .

Abstract A Saskatchewan multi-incident family was clinically characterized with Parkinson disease (PD) and Lewy body pathology. PD segregates as an autosomal-dominant trait, which could not be ascribed to any known mutation. DNA from three affected members was subjected to exome sequencing. Genome alignment, variant annotation and comparative analyses were used to identify shared coding mutations. Sanger sequencing was performed within the extended family and ethnically matched controls. Subsequent genotyping was performed in a multi-ethnic case–control series consisting of 2928 patients and 2676 control subjects from Canada, Norway, Taiwan, Tunisia, and the USA. A novel mutation in receptor-mediated endocytosis 8/RME-8 (DNAJC13 p.Asn855Ser) was found to segregate with disease. Screening of cases and controls identified four additional patients with the mutation, of which two had familial parkinsonism. All carriers shared an ancestral DNAJC13 p.Asn855Ser haplotype and claimed Dutch–German–Russian Mennonite heritage. DNAJC13 regulates the dynamics of clathrin coats on early endosomes. Cellular analysis shows that the mutation confers a toxic gain-of-function and impairs endosomal transport. DNAJC13 immunoreactivity was also noted within Lewy body inclusions. In late-onset disease which is most reminiscent of idiopathic PD subtle deficits in endosomal receptor-sorting/recycling are highlighted by the discovery of pathogenic mutations VPS35, LRRK2 and now DNAJC13. With this latest discovery, and from a neuronal perspective, a temporal and functional ecology is emerging that connects synaptic exo- and endocytosis, vesicular trafficking, endosomal recycling and the endo-lysosomal degradative pathway. Molecular deficits in these processes are genetically linked to the phenotypic spectrum of parkinsonism associated with Lewy body pathology.

Purpose Indications for sentinel lymph node biopsy (SLNB) for thin melanoma are continually evolving. We present a large multi-institutional study to determine factors predictive of sentinel lymph node (SLN) metastasis in thin melanoma. Patients and Methods Retrospective review of the Sentinel Lymph Node Working Group database from 1994 to 2012 identified 1,250 patients who had an SLNB and thin melanomas (≤ 1 mm). Clinicopathologic characteristics were correlated with SLN status and outcome. Results SLN metastases were detected in 65 (5.2%) of 1,250 patients. On univariable analysis, rates of Breslow thickness ≥ 0.75 mm, Clark level ≥ IV, ulceration, and absence of regression differed significantly between positive and negative SLN groups (all P < .05). These four variables and mitotic rate were used in multivariable analysis, which demonstrated that Breslow thickness ≥ 0.75 mm (P = .03), Clark level ≥ IV (P = .05), and ulceration (P = .01) significantly predicted SLN metastasis with 6.3%, 7.0%, and 11.6% of the patients with these respective characteristics having SLN disease. Melanomas < 0.75 mm had positive SLN rates of < 5% regardless of Clark level and ulceration status. Median follow-up was 2.6 years. Melanoma-specific survival was significantly worse for patients with positive versus negative SLNs (P = .001). Conclusion Breslow thickness ≥ 0.75 mm, Clark level ≥ IV, and ulceration significantly predict SLN disease in thin melanoma. Most SLN metastases (86.2%) occur in melanomas ≥ 0.75 mm, with 6.3% of these patients having SLN disease, whereas in melanomas < 0.75 mm, SLN metastasis rates are < 5%. By using a 5% metastasis risk threshold, SLNB is indicated for melanomas ≥ 0.75 mm, but further study is needed to define indications for SLNB in melanomas < 0.75 mm.

AbstractBackgroundBK virus (BKV)‐associated hemorrhagic cystitis (BKV‐HC) is a severe complication after hematopoietic stem cell transplantation (HSCT). Cidofovir (CDV) has emerged as an effective agent for the treatment of BKV nephropathy, but its use for BKV‐HC in pediatric HSCT recipients has not yet been established as a standard therapy.Patient and methodsWe retrospectively investigated the efficacy and safety of CDV therapy for patients with BKV‐HC at a single institution and analyzed the clinical management outcomes.ResultsFrom April 2009 to July 2011, 12 patients developed BKV‐HC at a median of 37 days after transplant (range 15–59 days). The cumulative incidence was 9% and the median peak of the urine BKV load was 2.5 × 1010 copies/mL (range 1.4 × 109–1.2 × 1011 copies/mL). Eleven patients received intravenous CDV (5 mg/kg/dose, with probenecid), whereas 1 patient received CDV (5 mg/kg/dose, without probenecid) intravesically. The median duration of therapy was 25 days (range 9–73 days), and a median of 2 doses was given (range 1–4). A reduction of ≥1 log in the BKV load was found in 11 patients, while 1 patient did not have any significant change in BKV load. Clinical improvement was observed in all cases, and no HC‐related death was observed. CDV‐related toxicity occurred in 1 patient (8%) and spontaneously resolved.ConclusionsCDV appears to be an effective and safe treatment for BKV‐HC in pediatric HSCT recipients, but prospective trials are warranted to support its use.

CTGF (connective tissue growth factor) is widely regarded as an important amplifier of the profibrogenic action of TGF-β (transforming growth factor β) in a variety of tissues, although the precise mechanism of how the TGF-β signalling pathways modulate CTGF expression remains unclear. In the present study, the role of PKCδ (protein kinase Cδ) in TGF-β1-mediated CTGF expression was investigated using HepG2 cells. TGF-β1 treatment specifically elevated PKCδ activation and CTGF expression. In contrast, blockade of PKCδ by the selective inhibitor Rottlerin or by siRNA knockdown significantly reduced TGF-β1-induced CTGF production. The regulatory mechanism was further demonstrated in HepG2 cells whereby TGF-β1-induced PKCδ activation negatively regulated the nuclear levels of PPM1A (protein phosphatase, Mg2+/Mn2+ dependent, 1A) through the RhoA/ROCK (Rho-associated kinase) pathway. Moreover, we showed that both Smad signalling and the PKCδ pathway appeared to be stimulated by TGF-β1 in parallel. Time course assessments indicated that PKCδ signalling may have a function in maintaining nuclear phospho-Smads at a maximal level. The collective results of the present study demonstrated that PKCδ-stimulated RhoA/ROCK activation resulted in a reduction in PPM1A, thereby up-regulating Smad-dependent gene induction for extended periods. These findings indicated that PKCδ plays a critical role in TGF-β1-induced CTGF production in HepG2 cells.