The regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as ...Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of
,
,
, and
(which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N
-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways.
A regulatory scheme was revealed in which AalR responds to l-Asp and DarR responds to d-Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi. Our studies improve understanding of transport and regulation and lay the foundation for determining how regulators distinguish l- and d-enantiomers. These studies are relevant for biotechnology applications, and they highlight the importance of d-amino acids as natural bacterial growth substrates.
Ampullary adenocarcinoma is a rare malignant neoplasm that arises within the duodenal ampullary complex. The role of adjuvant therapy (AT) in the treatment of ampullary adenocarcinoma has not been ...clearly defined.
To determine if long-term survival after curative-intent resection of ampullary adenocarcinoma may be improved by selection of patients for AT directed by histologic subtype.
This multinational, retrospective cohort study was conducted at 12 institutions from April 1, 2000, to July 31, 2017, among 357 patients with resected, nonmetastatic ampullary adenocarcinoma receiving surgery alone or AT. Cox proportional hazards regression was used to identify covariates associated with overall survival. The surgery alone and AT cohorts were matched 1:1 by propensity scores based on the likelihood of receiving AT or by survival hazard from Cox modeling. Overall survival was compared with Kaplan-Meier estimates.
Adjuvant chemotherapy (fluorouracil- or gemcitabine-based) with or without radiotherapy.
Overall survival.
A total of 357 patients (156 women and 201 men; median age, 65.8 years interquartile range, 58-74 years) underwent curative-intent resection of ampullary adenocarcinoma. Patients with intestinal subtype had a longer median overall survival compared with those with pancreatobiliary subtype (77 vs 54 months; P = .05). Histologic subtype was not associated with AT administration (intestinal, 52.9% 101 of 191; and pancreatobiliary, 59.5% 78 of 131; P = .24). Patients with pancreatobiliary histologic subtype most commonly received gemcitabine-based regimens (71.0% 22 of 31) or combinations of gemcitabine and fluorouracil (12.9% 4 of 31), whereas treatment of those with intestinal histologic subtype was more varied (fluorouracil, 50.0% 17 of 34; gemcitabine, 44.1% 15 of 34; P = .01). In the propensity score-matched cohort, AT was not associated with a survival benefit for either histologic subtype (intestinal: hazard ratio, 1.21; 95% CI, 0.67-2.16; P = .53; pancreatobiliary: hazard ratio, 1.35; 95% CI, 0.66-2.76; P = .41).
Adjuvant therapy was more frequently used in patients with poor prognostic factors but was not associated with demonstrable improvements in survival, regardless of tumor histologic subtype. The value of a multimodality regimen remains poorly defined.
Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of ...the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7R) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 x 10(-7)). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.
Pancreatic cysts are estimated to be present in 2%-3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive ...cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts.
We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 μL of cyst fluid.
We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity.
Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions.
.
Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of ...the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7R) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 x 10(-7)). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.
Abstract
Objectives
To develop a curriculum for a commercial reference laboratory clinical pathology training elective.
Methods
A 4-day elective at Quest Diagnostics was developed. The elective ...included 32 sessions composed of interactive didactic sessions and laboratory tours/demonstrations. Ten residents who attended the elective completed a written evaluation and scored each component of the curriculum.
Results
Written comments were very positive and demonstrated the goals of the elective were achieved. Laboratory tours and one-on-one sessions with the medical directors were especially well received. Most of the residents stated that the rotation gave them exposure to an area of laboratory medicine that they were not familiar with.
Conclusions
The elective provided a resident training experience that was highly regarded and exposed residents to an area of laboratory medicine not encountered in most pathology training programs. Our curriculum could serve as a model for establishing a similar elective in other training programs.
The aim of this study was to elucidate the impact of intraoperative blood loss on outcomes following pancreatoduodenectomy (PD).
The negative impact of intraoperative blood loss on outcomes in PD has ...long been suspected but not well characterized, particularly those factors that may be within surgeons' control.
From 2001 to 2015, 5323 PDs were performed by 62 surgeons from 17 institutions. Estimated blood loss (EBL) was discretized (0 to 300, 301 to 750, 751 to 1300, and >1300 mL) using optimal scaling methodology. Multivariable regression, adjusted for patient, surgeon, and institutional variables, was used to identify associations between EBL and perioperative outcomes. Factors associated with both increased and decreased EBL were elucidated. The relative impact of surgeon-modifiable contributors was estimated through beta coefficient standardization.
The median EBL of the series was 400 mL interquartile range (IQR) 250 to 600. Intra-, post-, and perioperative transfusion rates were 15.8%, 24.8%, and 37.2%, respectively. Progressive EBL zones correlated with intra- but not postoperative transfusion in a dose-dependent fashion (P < 0.001), with a key threshold of 750 mL EBL (8.14% vs 40.9%; P < 0.001). Increasing blood loss significantly correlated with poor perioperative outcomes. Factors associated with increased EBL were trans-anastomotic stent placement, neoadjuvant chemotherapy, pancreaticogastrostomy reconstruction, multiorgan or vascular resection, and elevated operative time, of which 38.7% of the relative impact was "potentially modifiable" by the surgeon. Conversely, female sex, small duct, soft gland, minimally invasive approach, pylorus-preservation, biological sealant use, and institutional volume (≥67/year) were associated with decreased EBL, of which 13.6% was potentially under the surgeon's influence.
Minimizing blood loss contributes to fewer intraoperative transfusions and better perioperative outcomes for PD. Improvements might be achieved by targeting modifiable factors that influence EBL.
Endothelin-1 is a vasoactive peptide that activates both the endothelin A (ET(A)) and endothelin B (ET(B)) receptors, and is secreted in high concentrations in many different cancer environments. ...Although ET(A) receptor activation has an established nociceptive effect in cancer models, the role of ET(B) receptors on cancer pain is controversial. EDNRB, the gene encoding the ET(B) receptor, has been shown to be hypermethylated and transcriptionally silenced in many different cancers. In this study we demonstrate that EDNRB is heavily methylated in human oral squamous cell carcinoma lesions, which are painful, but not methylated in human oral dysplasia lesions, which are typically not painful. ET(B) mRNA expression is reduced in the human oral squamous cell carcinoma lesions as a consequence of EDNRB hypermethylation. Using a mouse cancer pain model, we show that ET(B) receptor re-expression attenuates cancer-induced pain. These findings identify EDNRB methylation as a novel regulatory mechanism in cancer-induced pain and suggest that demethylation therapy targeted at the cancer microenvironment has the potential to thwart pain-producing mechanisms at the source, thus freeing patients of systemic analgesic toxicity.
Among neonatal cardiomyopathies, primary endocardial fibroelastosis (pEFE) remains a mysterious disease of the endomyocardium that is poorly genetically characterized, affecting 1/5000 live births ...and accounting for 25% of the entire pediatric dilated cardiomyopathy (DCM) with a devastating course and grave prognosis. To investigate the potential genetic contribution to pEFE, we performed integrative genomic analysis, using whole exome sequencing (WES) and RNA-seq in a female infant with confirmed pathological diagnosis of pEFE. Within regions of homozygosity in the proband genome, WES analysis revealed novel parent-transmitted homozygous mutations affecting three genes with known roles in cilia assembly or function. Among them, a novel homozygous variant c.1943delA of uncertain significance in
ALMS1
was prioritized for functional genomic and mechanistic analysis. Loss of function mutations of
ALMS1
have been implicated in Alstrom syndrome (AS) OMIM 203800, a rare recessive ciliopathy that has been associated with cardiomyopathy. The variant of interest results in a frameshift introducing a premature stop codon. RNA-seq of the proband’s dermal fibroblasts confirmed the impact of the novel
ALMS1
variant on RNA-seq reads and revealed dysregulated cellular signaling and function, including the induction of epithelial mesenchymal transition (EMT) and activation of TGFβ signaling. ALMS1 loss enhanced cellular migration in patient fibroblasts as well as neonatal cardiac fibroblasts, while ALMS1-depleted cardiomyocytes exhibited enhanced proliferation activity. Herein, we present the unique pathological features of pEFE compared to DCM and utilize integrated genomic analysis to elucidate the molecular impact of a novel mutation in
ALMS1
gene in an AS case. Our report provides insights into pEFE etiology and suggests, for the first time to our knowledge, ciliopathy as a potential underlying mechanism for this poorly understood and incurable form of neonatal cardiomyopathy.
Key message
Primary endocardial fibroelastosis (pEFE) is a rare form of neonatal cardiomyopathy that occurs in 1/5000 live births with significant consequences but unknown etiology.
Integrated genomics analysis (whole exome sequencing and RNA sequencing) elucidates novel genetic contribution to pEFE etiology.
In this case, the cardiac manifestation in Alstrom syndrome is pEFE. To our knowledge, this report provides the first evidence linking ciliopathy to pEFE etiology.
Infants with pEFE should be examined for syndromic features of Alstrom syndrome.
Our findings lead to a better understanding of the molecular mechanisms of pEFE, paving the way to potential diagnostic and therapeutic applications.
Working memory impairments are a core aspect of schizophrenia, yet current medicines do not address such cognitive dysfunction. We have developed a model of these working memory deficits by acutely ...disrupting glutamatergic synaptic transmission by administration of the
N-methyl-
d-aspartate (NMDA) antagonist ketamine in the nonhuman primate. The current studies evaluated the effect of positive allosteric modulators (“potentiators”) of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors on the working memory and behavioral effects of ketamine. AMPA receptors mediate fast excitatory synaptic transmission throughout the brain and play a critical role in the activity-dependent regulation of NMDA receptors. We find that positive modulation of AMPA receptors with LY451646 (0.1–1.0
mg/kg, SC) and structurally distinct PF-4778574 (0.01
mg/kg, SC) robustly ameliorates ketamine-induced working memory impairments without altering behavioral effects of acute ketamine we consider related to positive- and negative-like symptoms. These results support AMPA receptor potentiators as a potential adjunctive treatment for cognitive impairment associated with schizophrenia (CIAS).
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