Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation.

Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli.

While 20 canonical amino acids are used by most organisms for protein synthesis, the creation of cells that can use noncanonical amino acids (ncAAs) as additional protein building blocks holds great promise for preparing novel medicines and for studying complex questions in biological systems. However, only a small number of biosynthetic pathways for ncAAs have been reported to date, greatly restricting our ability to generate cells with ncAA building blocks. In this study, we report the creation of a completely autonomous bacterium that utilizes 3,4-dihydroxy-L-phenylalanine (DOPA) as its 21st amino acid building block. Like canonical amino acids, DOPA can be biosynthesized without exogenous addition and can be genetically incorporated into proteins in a site-specific manner. Equally important, the protein production yields of DOPA-containing proteins from these autonomous cells are greater than those from cells exogenously fed with 9 mM DOPA. The unique catechol moiety of DOPA can be used as a versatile handle for site-specific protein functionalizations via either oxidative coupling or strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition reactions. We further demonstrate the use of these autonomous cells in preparing fluorophore-labeled anti-human epidermal growth factor 2 (HER2) antibodies for the detection of HER2 expression on cancer cells.

Synthesis of precision antibody conjugates using proximity-induced chemistry.

Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.

Effect of wetland plants and bacterial inoculation on dissipation of phenanthrene.

This study attempts to evaluate the capacity of wetland plants' ability to dissipate phenanthrene (PHE) under waterlogged conditions. The results indicate that Typha latifolia and Vetiveria zizanioides may efficiently degrade PHE, and were much more effective when under combined plant cultivation with the inoculation of Pseudomonas frederiksbergensis (ATCC BAA-257) . Concentrations of PHE declined from 200 to less than 52 mg kg-1 in all treatments with plant cultivation. At the end of the experimental period, PHE was undetectable in combined plant cultivation in the presence of bacteria inoculation. Microbial biomass C(carbon), N(nitrogen), and P(phosphate) were significantly different (p < 0.05) in the presence and absence of bacteria inoculation with bacteria inoculation significantly (p < 0.05) increased microbial biomass P. The presence of bacteria inoculation and different plant species significantly (p < 0.05) decreased the PHE concentrations in the microcosms. The inoculation of bacteria and release of exudates from plant roots further enhanced the dissipation of PHE in sand. Concentrations of citric and malic acids were decreased up to 69% in bacteria-inoculated treatments, showing large citric and malic acids serving as a food source and growth substrate for bacteria.

Intermittent vibration protects aged muscle from mechanical and oxidative damage under prolonged compression.

Deep tissue pressure ulcers, a serious clinical challenge originating in the muscle layer, are hardly detectable at the beginning. The challenge apparently occurs in aged subjects more frequently. As the ulcer propagates to the skin surface, it becomes very difficult to manage and can lead to fatal complications. Preventive measures are thus highly desirable. Although the complex pathological mechanisms have not been fully understood, prolonged and excessive physical challenges and oxidative stress are believed to be involved in the ulcer development. Previous reports have demonstrated that oxidative stress could compromise the mechanical properties of muscle cells, making them easier to be damaged when physical challenges are introduced. In this study, we used senescence accelerated (SAMP8) mice and its control breed (SAMR1) to examine the protective effects of intermittent vibration on aged and control muscle tissues during prolonged epidermal compression under 100mmHg for 6h. Results showed that an application of 35Hz, 0.25g intermittent vibration during compression decreased the compression-induced muscle breakdown in SAMP8 mice, as indicated histologically in terms of number of interstitial nuclei. The fact that no significant difference in muscle damage could be established in the corresponding groups in SAMR1 mice suggests that SAMR1 mice could better accommodate the compression insult than SAMP8 mice. Compression-induced oxidative damage was successfully curbed using intermittent vibration in SAMP8 mice, as indicated by 8-OHdG. A possible explanation is that the anti-oxidative defense could be maintained with intermittent vibration during compression. This was supported by the expression level of PGC-1-alpha, catalase, Gpx-1 and SOD1. Our data suggested intermittent vibration could serve as a preventive measure for deep tissue ulcer, particularly in aged subjects.

Cortisol in human milk predicts child BMI.

OBJECTIVE: Breastfeeding has been linked to lower rates of childhood obesity. Human milk contains cortisol, known to regulate glucose storage and metabolism. The aim of this study was to to test the hypothesis that early exposure to cortisol in human breast milk helps to modulate infant body mass index (BMI) trajectories over the first 2 years of life. METHODS: Growth curve modeling was used to examine whether infant exposure to cortisol in human milk at 3 months predicted changes in child body mass index percentile (BMIP) at 6, 12, and 24 months of age in 51 breastfeeding mother-child pairs. RESULTS: Infants exposed to higher milk cortisol levels at 3 months were less likely to exhibit BMIP gains over the first 2 years of life, compared with infants exposed to lower milk cortisol. By age 2, infants exposed to higher milk cortisol levels had lower BMIPs than infants exposed to lower milk cortisol. Milk cortisol was a stronger predictor of BMIP change in girls than boys. CONCLUSIONS: Cortisol exposure through human milk may help to program metabolic functioning and childhood obesity risk. Further, because infant formula contains only trace amounts of glucocorticoids, these findings suggest that cortisol in milk is a novel biological pathway through which breastfeeding may protect against later obesity.

Histone deacetylase 6-mediated selective autophagy regulates COPD-associated cilia dysfunction.

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/- or Map1lc3B-/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6-/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1-/-, Map1lc3B-/-, and Hdac6-/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

Root exudates of wetland plants influenced by nutrient status and types of plant cultivation.

The present study investigated the amounts of root exudates and composition of organic acids released from two wetland plants (Typha latifolia and Vetiver zizanioides) under two nutrient treatments: low level (0.786 mM N and 0.032 mM P) and high level (7.86 mM N and 0.32 mM P) and two types of plant cultivation: monoculture and co-culture of the two plants. Low nutrient treatment significantly (p < 0.05) increased the root exudates of T. latifolia during the initial growth period (1-21 d) and those of V. zizanioides and the co-culture during the whole growth period. The concentrations of dissolved organic carbon in the root exudates of the co-culture in the low nutrient treatment were 3.23-7.91 times of those in the high nutrient treatment during the medium growth period (7-28 d). The compositions of organic acids varied between the two plant species and between the two nutrient treatments. The pattern of organic acids was also different between the co-culture and the monoculture. Oxalic acid was by far the major organic acid exuded from the two wetland plants. The present study on root exudates suggests that co-culture of wetland plant species would be more useful in the reclamation of waste water than a monoculture system.

Reliability of the Chinese version of the perceived family burden scale.

The study examined reliability of the 24-item Chinese version of the perceived family burden scale (CPFBS), which is a measure of behaviors associated with Chinese people with schizophrenia and the impacts of these behaviors on caregivers and relatives. Many patients with mental illness are returning to the community and living with families. Unfortunately, family members who have to live with the patients experience a sense of burden. The CPFBS is able to measure the perceived burden of the family, which enables therapists to formulate the most appropriate family intervention for them. The CPFBS was translated and culturally adapted from the English version. Some 21 main caregivers of individuals with schizophrenia were asked to rate the items on the scale by a competent administrator through a telephone interview (this was carried out twice, with 1 day between each interview). The results showed that CPFBS had high internal consistency (alpha=0.85) and acceptable test-retest reliability (r=0.86). We suggest that the CPFBS is ready to be used by clinicians and researchers in the study on family burden of individuals with schizophrenia in Hong Kong and in Chinese communities in other countries.