Quantifying the progression of non-alcoholic fatty liver disease in human biomimetic liver microphysiology systems with fluorescent protein biosensors.

Metabolic syndrome is a complex disease that involves multiple organ systems including a critical role for the liver. Non-alcoholic fatty liver disease (NAFLD) is a key component of the metabolic syndrome and fatty liver is linked to a range of metabolic dysfunctions that occur in approximately 25% of the population. A panel of experts recently agreed that the acronym, NAFLD, did not properly characterize this heterogeneous disease given the associated metabolic abnormalities such as type 2 diabetes mellitus (T2D), obesity, and hypertension. Therefore, metabolic dysfunction-associated fatty liver disease (MAFLD) has been proposed as the new term to cover the heterogeneity identified in the NAFLD patient population. Although many rodent models of NAFLD/NASH have been developed, they do not recapitulate the full disease spectrum in patients. Therefore, a platform has evolved initially focused on human biomimetic liver microphysiology systems that integrates fluorescent protein biosensors along with other key metrics, the microphysiology systems database, and quantitative systems pharmacology. Quantitative systems pharmacology is being applied to investigate the mechanisms of NAFLD/MAFLD progression to select molecular targets for fluorescent protein biosensors, to integrate computational and experimental methods to predict drugs for repurposing, and to facilitate novel drug development. Fluorescent protein biosensors are critical components of the platform since they enable monitoring of the pathophysiology of disease progression by defining and quantifying the temporal and spatial dynamics of protein functions in the biosensor cells, and serve as minimally invasive biomarkers of the physiological state of the microphysiology system experimental disease models. Here, we summarize the progress in developing human microphysiology system disease models of NAFLD/MAFLD from several laboratories, developing fluorescent protein biosensors to monitor and to measure NAFLD/MAFLD disease progression and implementation of quantitative systems pharmacology with the goal of repurposing drugs and guiding the creation of novel therapeutics.

Secure Closure of Right Ventricular Perforation Reinforced With Coronary Artery Wall.

Accidental entry into the right ventricular cavity is a common occurrence during exposure of the intra-myocardial left anterior descending artery. Several techniques have been described for repair of the perforation. Although these methods can be used, there is still a danger of persistent bleeding or distal ischemia. We describe a method that is safe and reproducible.

Multiparameter Analysis Identifies Heterogeneity in Knee Osteoarthritis Synovial Responses.

OBJECTIVE: Synovial membrane inflammation is common in osteoarthritis (OA) and increases cartilage injury. However, synovial fluid and histology studies suggest that OA inflammatory responses are not homogeneous. Greater understanding of these responses may provide new insights into OA disease mechanisms. We undertook this study to develop a novel multiparameter approach to phenotype synovial responses in knee OA. METHODS: Cell composition and soluble protein production were measured by flow cytometry and multiplex enzyme-linked immunosorbent assay in synovium collected from OA patients undergoing knee replacement surgery (n = 35). RESULTS: Testing disaggregation conditions showed that aggressive digestion improved synovial cell yield and mesenchymal staining by flow cytometry, but it negatively impacted CD4+ T cell and CD56+ natural killer cell staining. Less aggressive digestion preserved these markers and showed highly variable T cell infiltration (range 0-43%; n = 32). Correlation analysis identified mesenchymal subpopulations associated with different nonmesenchymal populations, including macrophages and T cells (CD45+CD11b+HLA-DR+ myeloid cells with PDPN+CD73+CD90-CD34- mesenchymal cells [r = 0.65, P < 0.0001]; and CD45+CD3+ T cells with PDPN+CD73+CD90+CD34+ mesenchymal cells [r = 0.50, P = 0.003]). Interleukin-6 (IL-6) measured by flow cytometry strongly correlated with IL-6 released by ex vivo culture of synovial tissue (r = 0.59, P = 0.0012) and was highest in mesenchymal cells coexpressing CD90 and CD34. IL-6, IL-8, complement factor D, and IL-10 release correlated positively with tissue cellularity (P = 0.0042, P = 0.018, P = 0.0012, and P = 0.038, respectively). Additionally, increased CD8+ T cell numbers correlated with retinol binding protein 4 (P = 0.033). Finally, combining flow cytometry and multiplex data identified patient clusters with different types of inflammatory responses. CONCLUSION: We used a novel approach to analyze OA synovium, identifying patient-specific inflammatory clusters. Our findings indicate that phenotyping synovial inflammation may provide new insights into OA patient heterogeneity and biomarker development.

Modified Infarct Exclusion Technique for Repair of Postinfarction Ventricular Septal Rupture.

Repair of ventricular septal rupture after acute myocardial infarction remains a surgical challenge. Several techniques for the closure of these defects have been described. This report discusses an infarct exclusion technique modified from the one described by Tirone David and associates. In this technique two separate pericardial patches are used. The first patch excludes the rupture. The second patch is sutured to the margins of the first patch and thus provides strength to the margins of the first patch. The repair is simple and durable and has reduced the incidence of residual ventricular septal defects and patch dehiscence.

High Keratin 8/18 Ratio Predicts Aggressive Hepatocellular Cancer Phenotype.

BACKGROUND & AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: We analyzed livers of aged Krt18-/- mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18-/- mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18-/- mice with 26 human hepatoma cell lines and with data sets of >300 patients with HCC, where Krt18-/- gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC.

Aortic transection with superior biatrial trans-septal approach for triple valve re-do procedures.

The repair of atrioventricular valves requires optimal valve exposure through even unconventional surgical approaches. This is especially true in the situation of multiple re-do valve surgeries, where extensive adhesions may prevent adequate valve exposure. When multiple valve surgeries are contemplated, an extensive incisional approach is required without causing damage to any surrounding crucial structures. Re-do surgeries are described in patients with prosthetic valve endocarditis where the correction of all valves involved is necessary, as is the correction of underlying ventricular pathologies such as sinuses and atrioventricular tunnels. As several surgical approaches have been attempted in these situations, a technique is described which involves a superior biatrial approach after transecting the aorta, which addresses the mitral, tricuspid and aortic valves and allows the correction of associated ventricular pathologies.

Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.

We focus on hepatic inclusions, which are defined as intracellular aggregates of stainable substances. They represent established hallmarks of their respective human disorders, but unlike aggregates found in neurodegenerative disorders are often not well studied. Hepatic inclusions can be subdivided into primary liver aggregates and aggregates found in multiple tissues. The former ones consist of inclusions found in endoplasmic reticulum storage diseases such as α 1-antitrypsin aggregates or ground-glass hepatocytes, p62-containing (Mallory-Denk bodies and intracellular hyaline bodies) and porphyrin-containing inclusions. p62-containing aggregates are not restricted to the liver but are found in multiple other disorders such as Parkinson or Alzheimer disease. Inclusions such as pale bodies or intracellular hyaline bodies are typical for malignant disorders while others (ground-glass hepatocytes and α1-antitrypsin aggregates) are predominantly seen in non-neoplastic tissues. The inclusions, which are not restricted to the liver, are often due to a systemic viral infection, but also due to disruption of glycogen metabolism or systemic inclusion-forming diseases such as polyglutamine disorders or sarcoidosis. Despite their heterogeneity, inclusions share several pathogenic principles such as an imbalance between protein damage/misfolding on one side and repair/degradation on the other side. This is why hepatic aggregates represent a valuable tool to study the aggregation process in general and to improve our understanding of inclusions found in multiple human disorders.

A simple technique for securing Gore-Tex neochordal height and knot slippage in mitral valve repair.

Polytetrafluoroethylene or Gore-Tex sutures are used for chordal replacement, and are durable but extremely slippery, a property which causes knot slippage and ineffective neochordal length. We report a modification that is simple and reproducible in preventing knot slippage and maintaining optimal neochordal length, without the use of devices or additional sutures.

Cross ß-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross ß-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross ß-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α1-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross ß-sheet conformation than K18, whereas p62 never had cross ß-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly α-helical to cross ß-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8⁻/⁻ mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

Bilateral chylothorax, chylopericardium and chylous ascitis.

Non-Hodgkin's lymphoma (NHL) can commonly present as chylothorax and rarely as chylopericardium. Here we are presenting a case of a 21-years-old female with bilateral chylothorax, chylopericardium and chylous ascites all together finally diagnosed to have NHL as the etiology. To the best of our knowledge, it has been reported very infrequently.

Rare vascular anomaly mimicking bronchogenic carcinoma.

We report a case of anomalous left pulmonary artery (pulmonary artery sling) detected incidentally on computed tomography thorax. This was carried out to rule out bronchogenic carcinoma in a patient of chronic obstructive pulmonary disease who presented with streaking. He was a chronic smoker having bilateral hilar prominence on chest radiograph.

Diffuse alveolar hemorrhage in Wegener's granulomatosis.

Diffuse alveolar hemorrhage is a life-threatening though rare manifestation of Wegener's granulomatosis (WG). An active diagnostic workup, intensive observation, and aggressive immunosuppressive treatment are cornerstones of the management. The treatment modalities available for such complications are pulse cyclophosphamide therapy with steroids. We report here a case of WG with diffuse alveolar hemorrhage as the first manifestation of the disease in life that responded to steroids and cyclophosphamide.

Pulmonary embolism in cases of pulmonary tuberculosis: a unique entity.

Pulmonary tuberculosis is very prevalent in developing countries but its thrombogenic potential is a new entity. There are reports stating the relation of Deep Vein Thrombosis (DVT) with severe forms of tuberculosis but no literature is available for correlation of pulmonary tuberculosis and pulmonary embolism. We are presenting series of five patients with different forms of tuberculosis presenting with pulmonary embolism having no risk factor for hypercoagulability. Also, serum protein C, protein S, antithrombin and factor V levels were normal in all. We are highlighting an unreported phenomenon so that high suspicion, adequate prophylaxis and prompt management of pulmonary embolism can play a vital role in the survival of this subset of patients.

Erythema nodosum: Atypical presentation of common disease.

Tuberculosis is a very common disease often presenting in an uncommon form. High level of suspicion is required to diagnose it, thereby preventing its morbidity and mortality. We present a case of young female with multiple tuberculo-protein hypersensitivity reactions without any evidence of active tuberculosis in the body.

Combined modality of mechanical breakdown and intraembolus thrombolysis in failed systemic thrombolysis of subacute pulmonary embolism patients.

OBJECTIVES: Acute massive pulmonary embolism with failed systemic thrombolysis has a high morbidity and mortality with few treatment options available. This study assesses the role of combined modality of mechanical fragmentation and intralesional thrombolysis in failed systemic thrombolysis. METHODS: Seven (13.5%) of 52 patients with massive pulmonary embolism with persistent cardiogenic shock that failed systemic thrombolysis underwent imaging to confirm pulmonary embolism, and then mechanical fragmentation and intralesional thrombolysis. Mechanical breakdown of embolus was accomplished with 5-F multipurpose catheter to re-establish flow, followed by intralesional infusion of urokinase (4,400 IU/kg over 10 minutes followed by 4,400 IU/kg per hour over 24 hours). RESULTS: Four (57.1%) of 7 were unsuccessfully thrombolyzed outside the hospital by urokinase, 2 (28.6%) with recombinant tissue-type plasminogen activator (rtPA) and 1 (14.3%) with streptokinase systemically before 24-48 hours of admission. At presentation, average heart rate and shock index were 121.7/min and 1.45, respectively. Average systolic pulmonary arterial pressure was 73 ± 2.65 mmHg at presentation, and postoperatively was significantly reduced to 39.7 ± 10.44 mmHg (P < 0.001). Mortality at 24 hours, 30 days, and 2 years follow-up was 0% (0/7). CONCLUSIONS: Mechanical breakdown of thrombus followed by urokinase infusion may be a cost-effective, minimally invasive, and potentially life-saving procedure for the management of acute massive pulmonary embolism. Randomized controlled trials are required to compare this new strategy to contemporary conventional approaches.

Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes.

BACKGROUND: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue. RESULTS: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi. CONCLUSION: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101.

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.

Tuberculous otitis media with postaural abscess and submandibular lymphadenopathy.

We are reporting a case of right-sided tuberculous otitis media with postaural abscess and multiple submandibular lymphadenopathy which has been reported very infrequently. A high level of suspicion by the treating physician is mandatory to avoid long delay in diagnosis and increased complications in the modern chemotherapy era.