Hepatopulmonary Syndrome in an Adolescent With Insidious Hypoxia and Small Intrahepatic Portal Venous Shunts: Posttransplant Benefit From Sildenafil.

We report a patient without known preexisting liver disease who presented with hepatopulmonary syndrome (HPS) due to aberrant intrahepatic portal venous development leading to portosystemic shunting. Liver transplantation resulted in resolution of portal hypertension and HPS and sildenafil was safely tolerated in the treatment of persistent fatigue and hypoxemia. Twelve months later, patient has normal allograft function and has returned to normal activity.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c.

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; -19 mV, M81A; -69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these "release mutants."

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag.

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope "tags" to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways in vitro. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and in vivo imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics.

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion.

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCPo (OCP at orange state) is converted into OCPr(OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCPr to OCPo, however, is largely delayed-almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu2+-locked OCPr. The Cu2+-OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.

Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP is the predominant state at protein concentrations ranging from 3 to 180 µM and that higher-order oligomers gradually form at protein concentrations above this range. The OCP, however, demonstrates significantly different oligomerization behavior. Monomeric OCP (mOCP) dominates at low protein concentrations, with an observable population of dimeric OCP (dOCP). The ratio of dOCP to mOCP, however, increases proportionally with protein concentration. Higher-order OCP oligomers form at protein concentrations beyond 10 µM. Additionally, native mass spectrometry coupled with ion mobility allowed us to measure protein collisional cross sections and interrogate the unfolding of different FRP and OCP oligomers. We found that monomeric FRP exhibits a one-stage unfolding process, which could be correlated with its C-terminal bent crystal structure. The structural domain compositions of FRP and OCP are compared and discussed.

Improving theranostics in pancreatic cancer.

BACKGROUND: Pancreatic cancer is the fourth most deadly cancer in the United States, and is expected to be the second most deadly by 2030. The major difficulty in treating pancreatic cancer is the late onset of symptoms. Generally, patients show metastatic disease by the time of diagnosis, with a survival rate of 5% beyond 5 years. In patients without metastatic disease, surgical resection increases 5 year survival rate to 25%. The remaining 75% succumb to undetected metastases. Clearly, improvements to both detection, surgical intervention, and therapeutic strategies will be needed to improve patient outcome in pancreatic cancer. METHODS: Recent literature has been surveyed and atomic models of new therapeutic approaches were generated. RESULTS AND CONCLUSIONS: Here, we focus on the recent progress employing monoclonal antibodies (mAbs) to target pancreatic cancer associated markers, and more specifically on recent chemical and protein engineering efforts to improve the homogeneity, stability, and administration of mAbs to precisely deliver imaging agents and cytotoxins to sites of disease.

Dramatic Domain Rearrangements of the Cyanobacterial Orange Carotenoid Protein upon Photoactivation.

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection.

Transcriptomic analysis illuminates genes involved in chlorophyll synthesis after nitrogen starvation in Acaryochloris sp. CCMEE 5410.

Acaryochloris species are a genus of cyanobacteria that utilize chlorophyll (chl) d as their primary chlorophyll molecule during oxygenic photosynthesis. Chl d allows Acaryochloris to harvest red-shifted light, which gives them the ability to live in filtered light environments that are depleted in visible light. Although genomes of multiple Acaryochloris species have been sequenced, their analysis has not revealed how chl d is synthesized. Here, we demonstrate that Acaryochloris sp. CCMEE 5410 cells undergo chlorosis by nitrogen depletion and exhibit robust regeneration of chl d by nitrogen repletion. We performed a time course RNA-Seq experiment to quantify global transcriptomic changes during chlorophyll recovery. We observed upregulation of numerous known chl biosynthesis genes and also identified an oxygenase gene with a similar transcriptional profile as these chl biosynthesis genes, suggesting its possible involvement in chl d biosynthesis. Moreover, our data suggest that multiple prochlorophyte chlorophyll-binding homologs are important during chlorophyll recovery, and light-independent chl synthesis genes are more dominant than the light-dependent gene at the transcription level. Transcriptomic characterization of this organism provides crucial clues toward mechanistic elucidation of chl d biosynthesis.

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation.

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the ß-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

Structural analysis of the homodimeric reaction center complex from the photosynthetic green sulfur bacterium Chlorobaculum tepidum.

The reaction center (RC) complex of the green sulfur bacterium Chlorobaculum tepidum is composed of the Fenna-Matthews-Olson antenna protein (FMO) and the reaction center core (RCC) complex. The RCC complex has four subunits: PscA, PscB, PscC, and PscD. We studied the FMO/RCC complex by chemically cross-linking the purified sample followed by biochemical and spectroscopic analysis. Blue-native gels showed that there were two types of FMO/RCC complexes, which are consistent with complexes with one copy of FMO per RCC and two copies of FMO per RCC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples after cross-linking showed that all five subunits of the RC can be linked by three different cross-linkers: bissulfosuccinimidyl suberate, disuccinimidyl suberate, and 3,3-dithiobis-sulfosuccinimidyl propionate. The interaction sites of the cross-linked complex were also studied using liquid chromatography coupled to tandem mass spectrometry. The results indicated that FMO, PscB, PscD, and part of PscA are exposed on the cytoplasmic side of the membrane. PscD helps stabilize FMO to the reaction center and may facilitate transfer of the electron from the RC to ferredoxin. The soluble domain of the heme-containing cytochrome subunit PscC and part of the core subunit PscA are located on the periplasmic side of the membrane. There is a close relationship between the periplasmic portions of PscA and PscC, which is needed for the efficient transfer of the electron between PscC and P840.

Alternative Complex III from phototrophic bacteria and its electron acceptor auracyanin.

Alternative Complex III (ACIII) is a multisubunit integral membrane protein electron transfer complex that is proposed to be an energy-conserving functional replacement for the bacterial cytochrome bc1 or b6f complexes. Clues to the structure and function of this novel complex come from its relation to other bacterial enzyme families. The ACIII complex has menaquinone: electron acceptor oxidoreductase activity and contains protein subunits with multiple Fe-S centers and c-type hemes. ACIII is found in a diverse group of bacteria, including both phototrophic and nonphototrophic taxa. In the phototrophic filamentous anoxygenic phototrophs, the electron acceptor is the small blue copper protein auracyanin instead of a soluble cytochrome. Recent work on ACIII and the copper protein auracyanin is reviewed with focus on the photosynthetic systems and potential electron transfer pathways and mechanisms. Taken together, the ACIII complexes constitute a unique system for photosynthetic electron transfer and energy conservation. This article is part of a Special Issue entitled: Respiratory Complex III and related bc complexes.

Site-directed mutagenesis of the highly perturbed copper site of auracyanin D.

Type-1 copper proteins participate in redox reactions and biological catalysis. Significant variation exists within the electronic structure of type-1 copper sites, producing both blue and green proteins. Classical, "blue" sites have been extensively studied, but "green" sites have been poorly characterized. We recently discovered a green copper protein, called auracyanin D. Here, we report a series of axial ligand mutations in auracyanin D, and characterize the resulting spectral and redox changes. The resulting mutants appear blue, green, and red and vary in redox potential from +56mV to +786mV. This is the largest change in redox potential to date for any type-1 center. We found that in this green protein, modifications of the axial ligand produce significantly larger changes than similar mutations in blue type-1 copper sites.

Ghrelin and obestatin levels in children with failure to thrive and obesity.

OBJECTIVES: Ghrelin and obestatin are 2 gastric hormones with opposite effects on food intake and body weight. We investigated plasma ghrelin and obestatin in children with failure to thrive (FTT) and obesity as compared with age-matched controls. METHODS: A total of 63 children were included in the study: 13 with FTT, 17 with obesity, and 33 age-matched controls. Children fasted for at least 8 hours before specimen collection. Both hormones were measured using commercially available enzyme immunoassay kits. RESULTS: Ghrelin and obestatin levels in children with FTT were not significantly different from that of the age-matched controls (P >0.05). In children with obesity, the total ghrelin levels were significantly lower (P = 0.0003) and the obestatin levels significantly higher (P = 0.029) compared with those in controls. In the control group, the fasting ghrelin level was significantly higher in the younger (<3 years) than in the older children (>3 years; P = 0.0004). Obestatin levels correlated positively with weight-for-age percentiles in the obese group (P = 0.011) and negatively in the control group >3 years (P = 0.019). CONCLUSIONS: Compared with the levels in age-matched controls, fasting ghrelin and obestatin levels did not differ significantly in children with FTT. In the children with obesity, the decreased ghrelin and increased obestatin levels suggest a possible adaptive process to positive energy balance. Ghrelin had pronounced age-related changes, and obestatin was associated with the weight status. This may suggest that these 2 hormones use different mechanisms to regulate energy balance and weight.

Metalloproteins diversified: the auracyanins are a family of cupredoxins that stretch the spectral and redox limits of blue copper proteins.

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display the entire known spectral and redox variations known in the type 1 copper site. C. aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon.

Haploinsufficiency of STK11 and neighboring genes cause a contiguous gene syndrome including Peutz-Jeghers phenotype.

We report on clinical and molecular findings of a 15-year-old female referred to our genetics clinic for a diagnostic evaluation due to mild developmental delay, submucosal cleft palate, and seizure disorder. Chromosomal microarray technology revealed a cancer predisposition due to a terminal deletion on chromosome 19p that includes the tumor suppressor gene STK11. In addition to abnormal lip pigmentation on exam, further diagnostic workup with upper and lower gastrointestinal screening confirmed polyps consistent with Peutz-Jeghers syndrome. The purpose of this study is to present a full clinical description of a patient with a rare 19p13.3 chromosomal deletion and review the current literature of this newly emerging contiguous gene deletion syndrome. It also supports the screening for complications of Peutz-Jeghers syndrome in all patients with this deletion.

Effect of length of controlled ovarian hyperstimulation using a gonadotropin-releasing hormone antagonist on in vitro fertilization pregnancy rates.

OBJECTIVE: To compare pregnancy outcomes between shorter and longer in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles using GnRH antagonist protocol. STUDY DESIGN: Retrospective cohort analysis at a large military academic hospital. A total of 351 patients underwent 412 IVF/ICSI cycles using a GnRH antagonist protocol from September 2002 through May 2008. Clinical pregnancy and live birth rates for all IVF/ICSI cycles were compared independently for both total length of ovarian stimulation with gonadotropins (< 10 days vs. > or = 10 days) and GnRH antagonist use (< 4 days vs. > or = 4 days), respectively. RESULTS: Clinical pregnancy rates were 54.6% among cycles with total gonadotropin use <10 days vs. 48.6% for those cycles > or = 10 days, odds ratio 0.82 (0.53-1.27); live birth rates were 50.0% vs. 47.7%, odds ratio 0.91 (0.59-1.42). Clinical pregnancy rates were 54.0% among cycles with GnRH antagonist use < 4 days vs. 52.8% with GnRH antagonist use > or = 4 days, odds ratio 0.95 (0.62-1.45); live birth rates were 46.8% vs. 50.4%, odds ratio 1.15 (0.76-1.76). CONCLUSION: Clinical pregnancy and live birth rates are not adversely affected by longer IVF/ICSI cycles using GnRH antagonists.

Deep Learning Algorithms for Diagnosis of Lung Cancer: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis of the diagnostic performance of current deep learning algorithms for the diagnosis of lung cancer. We searched major databases up to June 2022 to include studies that used artificial intelligence to diagnose lung cancer, using the histopathological analysis of true positive cases as a reference. The quality of the included studies was assessed independently by two authors based on the revised Quality Assessment of Diagnostic Accuracy Studies. Six studies were included in the analysis. The pooled sensitivity and specificity were 0.93 (95% CI 0.85−0.98) and 0.68 (95% CI 0.49−0.84), respectively. Despite the significantly high heterogeneity for sensitivity (I2 = 94%, p < 0.01) and specificity (I2 = 99%, p < 0.01), most of it was attributed to the threshold effect. The pooled SROC curve with a bivariate approach yielded an area under the curve (AUC) of 0.90 (95% CI 0.86 to 0.92). The DOR for the studies was 26.7 (95% CI 19.7−36.2) and heterogeneity was 3% (p = 0.40). In this systematic review and meta-analysis, we found that when using the summary point from the SROC, the pooled sensitivity and specificity of DL algorithms for the diagnosis of lung cancer were 93% and 68%, respectively.

Eosinophilic esophagitis: perspectives of adult and pediatric gastroenterologists.

To survey pediatric (PGI) and adult gastroenterologists (AGI) regarding their perceptions about the etiology, diagnosis, and management of eosinophilic esophagitis (EoE), and to assess whether differences in the clinical approach to EoE exist between these subspecialists. A 21-item survey related to EoE was emailed to PGI who subscribe to the PEDSGI Bulletin Board, and to two AGI per Electoral College vote in the US, randomly selected from each state. The survey was voluntary, and consent was assumed based on survey submission. The responses were submitted anonymously and results compiled in a secure Web site. A total of 249 physicians from across the globe responded to the survey, 68% of whom were PGI. The majority of respondents worked primarily in an academic institution or teaching hospital. Respondents revealed diagnosing an average of six cases (median 8, range 0-30) of EoE in the past 6 months. Ninety-two percent of AGI who see a patient with dysphagia and suspected EoE proceed to endoscopy with biopsies, compared to only 54% of PGI (P < 0.05); 38% of PGI would first perform an upper GI study. Both subspecialties agreed that biopsies of the proximal and distal esophagus are needed to make a definitive diagnosis of EoE. Fifty-eight percent PGI and 44% AGI defined EoE as an eosinophilic density of > or =20 per high power field (hpf) in esophageal biopsies. Seventy-seven percent of PGI but only 16% of AGI reported routine referral of patients for food allergy evaluation (P < 0.05). While 77% PGI and 91% of AGI would rely on a symptom-based follow-up, 27% PGI versus 9% AGI follow patients with biopsies according to a pre-determined schedule and another 38% repeat biopsies as needed, versus 15% AGI. This survey exposes a few inconsistencies among gastroenterologists in the diagnosis, management, and follow-up of patients with EoE. The currently available practice guidelines for the diagnosis and management of EoE are largely based on retrospective studies and expert opinion. The results of this survey suggest that a collaborative effort based on robust research is required upon us to develop evidence for how we care for these patients.

Modified Young-Dees-Leadbetter bladder neck reconstruction after exstrophy repair.

PURPOSE: We describe the application and results of modified Young-Dees-Leadbetter bladder neck reconstruction after successful complete primary repair in the newborn period. MATERIALS AND METHODS: The records of 34 patients referred for a continence procedure after successful exstrophy closure were extracted from an institutionally approved database. Patient characteristics and surgical outcomes were assessed. RESULTS: A total of 31 male and 3 female patients were identified, of whom 27 and 1, respectively, underwent osteotomy at initial closure. No patients attained urinary continence and so they were referred for a continence procedure. Nine patients did not have adequate bladder capacity for bladder neck repair (mean bladder capacity 63 ml, range 20 to 80). In those with suitable capacity mean capacity was 119 ml (range 85 to 180) and they underwent bladder neck reconstruction at a mean age of 4.9 years. Of the 25 patients who underwent bladder neck repair 14 (56%) were dry during the day and night, 5 (20%) were dry during the day but wet at night and 6 (24%) were totally incontinent. Pelvic osteotomies were performed at initial closure in 14 totally continent patients (100%) and in 4 (80%) with daytime continence but in no totally incontinent patients. All continent patients underwent hypospadias repair before age 1 year and none required ureteral reimplantation before bladder neck repair. CONCLUSIONS: A number of patients require bladder neck reconstruction to achieve continence after successful initial closure with complete primary repair. The modified Young-Dees-Leadbetter technique provides reasonable results with daytime and nighttime dryness attained by more than half of the patients.

Refining the Quality of Monoclonal Antibodies: Grafting Unique Peptide-Binding Site in the Fab Framework.

Monoclonal antibodies (mAbs) are a major therapeutic modality. Grafting the meditope binding site onto mAbs, also known as meditope-enabling, can extend the usefulness of mAbs by providing an additional protein-protein interaction surface without altering the stability or antigen binding. We have previously used this site for attaching dyes, cytotoxic drugs, and entire proteins. Here, we provide a simple protocol for meditope-enabling mAbs, and verifying meditope and antigen binding using flow cytometry (FACS).