Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Patterns and kinetics of hepatocellular carcinoma relapse post-liver transplantation: oligorecurrence and role of local therapies.

Background: Amongst patients with recurrent hepatocellular carcinoma (HCC) post-liver transplantation, systemic therapy options may be limited by immunosuppression or poor performance status. Thus, we aimed to assess the impact of metastasis-directed therapy to all sites of disease (MDT-All) in HCC patients with limited disease recurrence [i.e., oligorecurrence (oligoM1)] post-transplantation and characterize pre-transplant characteristics associated with oligoM1. Methods: In this retrospective cohort study, patients at a single institution with recurrent HCC post-liver transplantation were identified. OligoM1 disease was defined as ≤3 lesions at recurrence, while polyrecurrent (polyM1) disease was defined as >3 lesions. Outcomes were compared in patients with oligoM1 disease by receipt of MDT-All. Regression analyses were used to identify predictors of polyM1 disease and characteristics associated with post-recurrence outcomes. Results: Forty-three patients with recurrent HCC post-liver transplantation from 2005-2022 were identified. Twenty-seven (63%) patients had oligoM1. Microvascular invasion was independently associated with polyM1 [odds ratio (OR): 14.64; 95% confidence interval (CI): 1.48-144.77; P=0.022]. Elevated alpha-fetoprotein (AFP) ≥400 ng/mL [hazard ratio (HR): 2.44; 95% CI: 1.08, 5.52; P=0.033] at recurrence was independently associated with inferior overall survival (OS), while oligoM1 (HR: 0.42; 95% CI: 0.21, 0.87; P=0.018) was independently associated with favorable OS. Amongst patients with oligoM1 who received MDT-All (n=15) median OS was 38.4 vs. 16.1 months for those who did not receive MDT-All (log-rank P=0.021). There was a non-significant improvement in polyprogression-free survival (polyPFS) (median 14.0 vs. 10.7 months, P=0.1) amongst oligoM1 patients who received MDT-All compared to those who did not. Conclusions: Receipt of MDT-All was associated with improved OS amongst patients with limited HCC disease recurrence following liver transplantation.

Liver flukes diagnosed by ERCP in a local immigrant community.

BACKGROUND AND AIMS: Although a common pathogen in much of Asia, liver flukes are believed to be a rare cause of disease in the United States. In this series, we describe 3 patients diagnosed with Clonorchis sinensis during ERCP within 1 year at our institution. METHODS: Three patients referred to a large community hospital underwent ERCP with direct visualization of a worm in the biliary tree and subsequent histopathologic confirmation. RESULTS: The patients had variable clinical presentations, and 2 had repeat negative stool studies for ova and parasites. Each patient had imaging studies showing abnormalities within the biliary tree, after which ERCP was performed with direct visualization and extraction of a wormlike structure. It was confirmed that all 3 patients had emigrated from China within the last decade. The epidemiologic data and the histopathologic characteristics of the fluke eggs in utero were consistent with a diagnosis of C sinensis. CONCLUSIONS: The diagnosis of clonorchiasis should remain on the differential diagnosis for patients with nonspecific biliary symptoms who have known risk factors for this uncommonly common pathogen.

PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis.

PNC-27, a 32-residue peptide that contains an HDM-2 binding domain and a cell-penetrating peptide (CPP) leader sequence kills cancer, but not normal, cells by binding to HDM-2 associated with the plasma membrane and induces the formation of pores causing tumor cell lysis and necrosis. Conformational energy calculations on the structure of PNC-27 bound to HDM-2 suggest that 1:1 complexes form between PNC-27 and HDM-2 with the leader sequence pointing away from the complex. Immuno-scanning electron microscopy was carried out with cancer cells treated with PNC-27 and decorated with an anti-PNC-27 antibody coupled to 6 nm gold particles and an anti-HDM-2 antibody linked to 15 nm gold particles. We found multiple 6 nm- and 15 nm-labeled gold particles in approximately 1:1 ratios in layered ring-shaped structures in the pores near the cell surface suggesting that these complexes are important to the pore structure. No pores formed in the control, PNC-27-treated untransformed fibroblasts. Based on the theoretical and immuno-EM studies, we propose that the pores are lined by PNC-27 bound to HDM-2 at the membrane surface with the PNC-27 leader sequence lining the pores or by PNC-27 bound to HDM-2.

Mortality Rate and Predictors Among Patients with COVID-19 Related Acute Respiratory Failure Requiring Mechanical Ventilation: a Retrospective Single Centre Study.

AIM: The objective of the study was to assess mortality rates in COVID-19 patients suffering from acute respiratory distress syndrome (ARDS) who also requiring mechanical ventilation. The predictors of mortality in this cohort were analysed, and the clinical characteristics recorded. MATERIAL AND METHOD: A single centre retrospective study was conducted on all COVID-19 patients admitted to the intensive care unit of the Epicura Hospital Center, Province of Hainaut, Belgium, between March 1st and April 30th 2020. RESULTS: Forty-nine patients were included in the study of which thirty-four were male, and fifteen were female. The mean (SD) age was 68.8 (10.6) and 69.5 (12.6) for males and females, respectively. The median time to death after the onset of symptoms was eighteen days. The median time to death, after hospital admission was nine days. By the end of the thirty days follow-up, twenty-seven patients (55%) had died, and twenty-two (45%) had survived. Non-survivors, as compared to those who survived, were similar in gender, prescribed medications, COVID-19 symptoms, with similar laboratory test results. They were significantly older (p = 0.007), with a higher co-morbidity burden (p = 0.026) and underwent significantly less tra-cheostomy (p < 0.001). In multivariable logistic regression analysis, no parameter significantly predicted mortality. CONCLUSIONS: This study reported a mortality rate of 55% in critically ill COVID-19 patients with ARDS who also required mechanical ventilation. The results corroborate previous findings that older and more comorbid patients represent the population at most risk of a poor outcome in this setting.

Pseudo-acute kidney injury after minor trauma: A case report and review of literature.

Acute kidney injury (AKI), a common diagnosis in the emergency department, is defined as a reduction in renal filtration function, with decrease in urine output, increase in serum creatinine, or both. However, a rise in serum creatinine can occur without AKI: the principal cause of a pseudo-AKI is urinary ascites, caused by urinary tract rupture, followed by reverse intraperitoneal dialysis and resorption of creatinine. The intraperitoneal leak of free urine is mainly traumatic, and half of the cases are iatrogenic. A case of intraperitoneal bladder rupture after minor trauma is presented with a review of the pathology of pseudo-AKI.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood-brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [ß-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, ß-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and ß-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.