Predictors of Short-term Mortality in Patients of Cirrhosis of Liver Presenting as Acute Kidney Injury: An In-hospital Prospective Observational Study.

Background: Acute kidney injury (AKI) is known to be associated with increased short-term mortality among cirrhotic patients. On this background, we designed this study to evaluate various causes of AKI among admitted patients with cirrhosis of liver and predictors of 90-day mortality. Methods: One hundred and two consecutive adult patients with cirrhosis of liver with AKI hospitalized between November 2016 and March 2018 were enrolled in this prospective study. Their detailed clinical profile, including biochemical parameters, the etiology of AKI, and their clinical outcome of survival or mortality at 90-days, were recorded. Results: The most common causes of AKI were infections, followed by hypovolemia, seen in 55.88% and 31.37% of the patients, respectively. Hepatorenal syndrome (HRS) was seen in 10.78%, while parenchymal renal disease was the least common (1.9%). The in-hospital mortality rate was 28.4%, while 90-day mortality was 39.21%. The HRS group had a high 90-day mortality rate of 54.54%. ROC analysis of various biochemical parameters revealed that serum creatinine (sCr), Model for End-Stage Liver Disease (MELD), International Normalized Ratio (INR), and Neutrophil-Lymphocyte ratio (NLR), followed by Child Turcotte Pugh (CTP), had high area under the curves of 0.785, 0.773, 0.747, 0.740, and 0.718, respectively, for the prediction of 90-day mortality. Conclusion: Infection is the commonest cause of AKI in cirrhosis; however, mortality in patients with HRS-AKI is higher than that in those with infection-related AKI. Serum creatinine at admission, INR, NLR, and CTP scores predict short-term mortality among patients with AKI in cirrhosis. Further, large prospective studies are needed to confirm these findings.

Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping.

Background and Objectives: Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1-10 on the chromosome 4-permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8-20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1, DNMT3B, or LRIF1. We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2. Methods: OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot-based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1, followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software. Results: We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%. Discussion: A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.

Testing for Facioscapulohumeral Muscular Dystrophy with Optical Genome Mapping.

The introduction of optical genome mapping has improved time constraints and a lack of specificity from previous methodologies when performing genome-wide analyses of samples. Optical genome mapping allows for the detection of structural variations, aberrations, and functionality traits from a single stained molecule of DNA. Though the preparation time is increased compared to previously utilized visualization techniques, optical genome mapping significantly reduces the time needed for analysis. Specifically, individual disease pipelines have been developed to rapidly analyze prepared samples. One of these diseases, Facioscapulohumeral Muscular Dystrophy (FSHD), is detected through quantification of the D4Z4 repeat array on chromosome 4q35. Optical genome mapping, with the ability to enumerate the repeats of the D4Z4 array, has demonstrated the capability to precisely diagnose FSHD. In this protocol, the preparation of samples and subsequent loading and analysis in an optical genome mapping system is discussed for the detection and analysis of FSHD. These methods should prove highly useful in FSHD analyses and beyond with the development of further disease analysis pipelines within the instrument. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Genomic DNA isolation, labeling, and staining Basic Protocol 2: Mapping and analysis with the Bionano Saphyr® system.

Treatment of Hospital-Acquired Infections in Patients with Cirrhosis - New Challenges.

Background: Hospital acquired infections (HAI) in the cirrhotic patients contribute to hepatic decompensation. With emergence of bacterial drug resistance, designing the treatment protocol of HA infection has become the foremost challenge. Purpose: To analyze the resistance pattern of organisms isolated from hospital-acquired (HA) infections and determine appropriate antibiotics treatment protocols for these infections. Study Design: A prospective hospital based observational study was undertaken. Patients and Methods: The present study was conducted over 18 months at Kasturba Medical College, Mangalore, Karnataka, India. Patients with suspected HA infections were subjected to clinical, hematological and microbiological evaluation. Antibiotic sensitivity evaluation was undertaken for the bacteria isolated from these patients. Results: During the study period, 398 patients with cirrhosis were 472 times admitted to the hospital for treatment. Out of these patients, 40 patients were diagnosed with 50 HA infections. Fifty five different organisms were isolated from these infections. It was found that these 55 bacteria isolates comprised 30 (54.54%) gram-negative (GN) and 25 (45.45%) gram-positive (GP) bacteria. Quite seriously, extended-spectrum beta-lactamase (ESBL) producers and methicillin-resistant Staphylococcus aureus (MRSA) were detected in 40% and 58% of GN and GP infections respectively. A total of 36 (65.4%) and (14.5%) 8 out of 55 isolated organisms exhibited multi-drug resistance (MDR) and extensive drug resistance (XDR) behavior, respectively. Conclusion: Cirrhosis patients with HA infection possess higher prevalence of MDR and XDR infections. In such sick patients, cephalosporin and quinolones are not the appropriate empirical antibiotics. Herein, we propose a tigecycline with carbapenem like meropenem and vancomycin based empirical antibiotics protocol to be prescribed for such patients. De-escalation is advised after the culture sensitivity report is obtained.

Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology.

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35-100.00%); 100.00% sensitivity for T18 (71.51-100.00%); and 100.00% sensitivity for T13 analyses (66.37-100.00%). The specificities were >99% for each trisomy (99.7% (99.01-99.97%) for T21; 99.5% (98.62-99.85%) for T18; 99.7% (99.03-99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.

A single NGS-based assay covering the entire genomic sequence of the DMD gene facilitates diagnostic and newborn screening confirmatory testing.

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two-tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time-consuming and expensive. We have developed a comprehensive next-generation sequencing (NGS)-based single-step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single-exon or multi-exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full-sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD.

Comparative Analysis of Change in pH, Oral Health Status, and the Count of Streptococcus mutans and Lactobacillus Species in the Oral Cavity in Patients with Gastroenteral Diseases Using Saliva: A Pilot Study.

BACKGROUND: Seventy million people are affected by gastroenteral (GI) disturbances throughout the world. Oral cavity possesses various bacteria that remain as healthy commensals or turn pathogenic due to shift of balance with disturbances in health, which is reflected in the oral cavity too. Studies have shown a possible oro-systemic link. This study aimed at assessing the effect of GI disease on oral health comparing levels of pH, microbiological counts, and oral health status between test and control groups. MATERIALS AND METHODS: This pilot study consisted of two groups: test group containing 14 participants (GI disease) and control group (healthy) containing 3 participants. Two saliva samples were collected per patient. One sample was inoculated onto Mitis Salivarius and Rugose agar plates at 37oC in the CO2 incubator for 2 days. The second sample was used for recording pH. Parameters such as decayed, missing, and filled teeth, plaque index, gingival index, probing pocket depth, and clinical loss of attachment were also recorded. The results were analyzed using Statistical Package for Social Sciences (SPSS) version 11.5. Regression analysis was applied to predict the three-microbe culture based on the pH and GI disease. RESULTS: The oral health parameters showed a higher number of missing teeth, higher bleeding on probing, higher values of plaque and gingival index, a higher amount of clinical loss of attachment, and acidic pH of saliva in the test group. Microbiological analysis showed more Streptococcus mutans in the control group (7,500-10,000 cfu/mL), with a mean of 8,833.33±1,258.31 cfu/mL; S. salivarius was more in the test group (2,000-25,000 cfu/mL) with a mean of 15,866.67±6,697.76 cfu/mL. Candida was seen only in the test group (2,166.67±2,549.51 cfu/mL) and absent in the control group. Lactobacillus was absent in both the groups. CONCLUSION: The present study suggests the relation between oral health and GI diseases. Hence, saliva could be used as an easy, non-invasive biomarker to analyze the gastroenteric status of the patient.



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Low-Pass Genome Sequencing: Validation and Diagnostic Utility from 409 Clinical Cases of Low-Pass Genome Sequencing for the Detection of Copy Number Variants to Replace Constitutional Microarray.

DNA copy number variants (CNVs) account for approximately 300 Mb of sequence variation in the normal human genome. Significant numbers of pathogenic CNVs contribute toward human genetic disorders. Recent studies suggest a higher diagnostic and clinical significance of low-pass genome sequencing (LP-GS) compared with chromosomal microarrays (CMAs). The performance metrics of the 5X LP-GS was compared with CMA to validate a low-cost and high-throughput method. LP-GS test performed on 409 samples (including 78 validation and 331 clinical) was evaluated using American College of Medical Genetics and Genomics guidelines. The CNV accuracy, precision, specificity, and sensitivity were calculated to be 100% for all previously characterized CNVs by CMA. Samples (n = 6) run at both approximately 30X GS and approximately 5X GS (LP-GS) average depth detected a concordance of 89.43% to 91.8% and 77.42% to 89.86% for overall single-nucleotide variants and insertions/deletions, respectively. In the 331 clinical samples, 17.2% each were classified as pathogenic/likely pathogenic and uncertain clinical significance. In addition, several cases with pathogenic CNVs were detected that were missed by CMA. This study demonstrates that LP-GS (5X GS) was able to reliably detect absence of heterozygosity, microdeletion/microduplication syndromes, and intragenic CNVs with higher coverage and resolution over the genome. Because of lower cost, higher resolution, and greater sensitivity of this test, our study in combination with other reports could be used in an evidence-based review by professional societies to recommend replacing CMAs.

Evaluation of p53 protein expression in Barrett esophagus.

BACKGROUND: Loss of heterozygosity of p53 along with aneuploidy is deemed to be the early molecular steps in Barrett metaplasia-dysplasia-adenocarcinoma sequence. Objective biomarkers need to be used along with microscopy for risk stratification to predict the progression of Barrett esophagus (BE) to carcinoma. AIM: This study aims to study p53 protein expression in dysplasia and correlate the same with morphology in BE. MATERIALS AND METHODS: A time-bound study was conducted from January 2011 to June 2015. All esophageal biopsies showing histological evidence of columnar epithelium with the presence of goblet cells were included. The cases which showed dysplasia were graded on hematoxylin and eosin stain. Evaluation of p53 immunohistochemistry staining was done on all the cases of BE. Dysplasia was correlated with the expression of p53 using Chi-square value (χ2) and Fischer's exact test wherever appropriate. P < 0.05 was considered to be statistically significant. RESULTS: Of 829 esophageal biopsies received, 119 were endoscopically suspected to be BE, of which 85 cases were confirmed on microscopy. In our study, there were 75 cases negative for dysplasia (88.2%), 8 with low-grade dysplasia (LGD) (9.4%), and two with high-grade dysplasia (HGD) (2.4%). Three cases of BE had associated adenocarcinoma. Immunostaining with p53 done on all the 85 cases showed positive staining in all cases with LGD, one with HGD and two with adenocarcinoma. In the present study, immunostaining with p53 showed 90% sensitivity, 89.3% specificity, positive predictive value of 52.9%, and negative predictive value of 98.5%. CONCLUSION: The technical simplicity, easy availability, and comparatively lower cost enhance the role of p53 as a biomarker in risk stratification for patients with BE.

Role of biliary tract cytology in the evaluation of extrahepatic cholestatic jaundice.

BACKGROUND: Endoscopic evaluation is critical in assessing the cause of obstructive jaundice. Cytological techniques including bile aspiration and biliary brushings have become the initial diagnostic modality. AIM: The aim of this study is to evaluate the role of endoscopic biliary tract cytology as a diagnostic tool in the evaluation of extrahepatic cholestatic jaundice. MATERIALS AND METHODS: A total of 56 biliary tract specimens including 34 bile aspirations and 22 biliary brushings from 41 consecutive patients who had presented with obstructive jaundice and underwent endoscopic retrograde cholangiopancreatography (ERCP) were assessed by cytological examination. The smears prepared were analyzed for standard cytological features. RESULTS: Cytologic diagnosis was adenocarcinoma in 13 (31.7%) cases, atypical in 2 (4.9%), reactive in 3 (7.3%) and benign changes in 19 (46.3%) cases. 4 (9.8%) cases were non-diagnostic. Serum bilirubin was significantly elevated in the malignant group. Biliary stricture was the most common finding on ERCP (68.3%). On cytological examination, presence of solitary, intact atypical cells, enlarged nuclei, irregular nuclear membrane, coarse chromatin and nucleoli were important cytologic criteria for differentiating malignant from benign biliary specimens. CONCLUSIONS: Regular use of bile cytology and brushings during ERCP evaluation of extrahepatic cholestatic jaundice is invaluable in obtaining a morphologic diagnosis. A systematic approach, use of strict cytomorphologic criteria and inclusion of significant atypia as malignant diagnosis may improve the sensitivity.

Symptomatic Morgagni's hernia in an elderly patient.

Hernia of Morgagni occurs through an anterior defect in the diaphragm. Symptoms of these hernias are attributable to the herniated viscera. In our case, there was partial obstruction due to herniation of the distal stomach and pylorus into the right hemithorax that was reduced surgically through a right thoracolapaorotomy. Of special emphasis are the various modalities used to diagnose this condition in our case.

Treatment parameters modulating regression of human melanoma xenografts by an antibody-drug conjugate (CR011-vcMMAE) targeting GPNMB.

PURPOSE: To investigate the pharmacological properties of the CR011-vcMMAE fully human antibody-drug conjugate (ADC), such as dose titrations, quantitation of the time (days) to complete regression, pharmacokinetics, and schedule dependency. Our prior study characterized a fully human antibody to GPNMB covalently linked to monomethylauristatin E, CR011-vcMMAE, and further demonstrated cell surface staining of melanoma lines susceptible to the immunoconjugate's cytotoxicity (Clin Cancer Res 2005; 12(4): 1373-1382). METHODS: The human SK-MEL-2 and SK-MEL-5 melanoma xenografts were used in athymic mice to assess anti-tumor efficacy. After s.c. implantation, tumors became established (60-100 mg), and treatment commenced by i.v. injection of the immunoconjugate or vinblastine or paclitaxel. Short-term anti-tumor effects (inhibition of tumor growth) and long-term effects (complete regression) were observed. RESULTS: CR011-vcMMAE induced regression of established human SK-MEL-2 and SK-MEL-5 xenografts at doses from 1.25 to 80 mg/kg treatment when administered intravenously every 4 days (4 treatments); strikingly, regressions were not associated with re-growth during the observation period (200 days). The disappearance rate of implants was dose dependent (minimum time, 18.5 days). Detectable serum CR011-vcMMAE >or=1 microg/mL (approximately 0.01 microM) was observed for >30 days post-dose; CR011-vcMMAE showed an elimination half-life of 10.3 days. A low volume of distribution suggested that CR011-vcMMAE was confined to blood and interstitial fluid. CR011-vcMMAE could be delivered by either a single bolus dose or by intermittent dosing (i.e., every 1, 2, 4, 8, or 16 days) with no discernible differences in the proportion of tumor-free survivors, indicating a lack of schedule dependency. The antibody-drug conjugate produced complete regressions, but the equivalent doses of free monomethylauristatin E or unconjugated antibody did not show anti-tumor effects. In addition, decreases in plasma tumor-derived human interleukin-8 coincided with tumor nodule disappearance. CONCLUSIONS: Short-term anti-tumor effects and long-term effects (complete regression) were observed with CR011-vcMMAE, but not with the reference agents. These results suggest that CR011-vcMMAE may provide therapeutic benefit in malignant melanoma.

Inhibition of in vitro and in vivo T cell responses by recombinant human Tim-1 extracellular domain proteins.

Members of the T cell, Ig domain and mucin domain (Tim) family of proteins have recently been implicated in the control of T cell-mediated immune responses. Tim-1 (HUGO designation HAVCR1) polymorphisms have been linked to the regulation of atopy in mice and humans, suggestive of a role in immune regulation. Tim-1 is expressed upon activation of T cells. In concert with the increased expression of Tim-1, a binding partner for the extracellular domain of Tim-1 (eTim-1) was induced on activated T cells, and mRNA expression data was consistent with the binding partner being Tim-4. We found that co-immobilized recombinant eTim-1 was able to inhibit T cell activation mediated by CD3 + CD28 mAb. eTim-1 mediated its inhibitory effects on proliferation by arresting cell cycle at G(0)/G(1) phase through regulation of cell cycle proteins. In vivo, administration of eTim-1 proteins led to a decrease in both ear (contact hypersensitivity to oxazolone) and joint (methylated BSA antigen-induced arthritis) swelling. The inhibitory activity of eTim-1 in the T(h)1-dependent models was evidence that eTim-1 is able to modulate T cell responses. Manipulation of the Tim-1 interaction with its binding partner on T cells may therefore provide a novel target for therapeutic intervention in T cell-mediated diseases.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

Recombinant semaphorin 6A-1 ectodomain inhibits in vivo growth factor and tumor cell line-induced angiogenesis.

The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class 6 Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class 6 Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent nontumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assays both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.

Electrospun fibers from wheat protein: investigation of the interplay between molecular structure and the fluid dynamics of the electrospinning process.

In the present work, we demonstrate the ability to electrospin wheat gluten, a polydisperse plant protein polymer that is currently available at roughly 0.50 dollars/lb. A variety of electrospinning experiments were carried out with wheat gluten from two sources, at different solution concentrations, and with native and denatured wheat gluten to illustrate the interplay between protein structure and the fluid dynamics of the electrospinning process. The presence of both cylindrical and flat fibers was observed in the nonwoven mats, which were characterized using both polarized optical microscopy and field emission scanning electron microscopy. Retardance images obtained by polarized optical microscopy exhibited evidence of molecular orientation at the surface of the fibers. We believe that fiber formation by electrospinning is a result of both chain entanglements and the presence of reversible junctions in the protein, in particular, the breaking and re-forming of disulfide bonds that occur via a thiol/disulfide interchange reaction. The presence of the highest molecular weight glutenin polymer chains in the wheat protein appeared to be responsible for the lower threshold concentration for fiber formation, relative to that of a lower molecular weight fraction of wheat protein devoid of the high molecular weight glutenin component. Denaturation of the wheat protein, however, clearly disrupted this delicate balance of properties in the experimental regimes we investigated, as electrospun fibers from the denatured state were not observed.

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.